Composite antibodies of human subgroup IV light chain capable of binding to tag-72

ABSTRACT

This invention concerns a subset of composite Hum4 V L , V H  antibodies with high affinities to a high molecular weight, tumor-associated sialylated glycoprotein antigen (TAG-72) of human origin. These antibodies have variable regions with (1) V L  segments derived from the human subgroup IV germline gene, and (2) a V H  segment which is capable of combining with the V L  to form a three dimensional structure having the ability to bind TAG-72. In vivo methods of treatment and diagnostic assay using these composite antibodies is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation-in-Part of application Ser. No.07/964,536 filed Oct. 20, 1992 now abandoned; and a Continuation-in-Partof application Ser. No. 07/510,697, filed Apr. 19, 1990, now abandoned.

FIELD OF THE INVENTION

The present invention is directed to the fields of immunology andgenetic engineering.

BACKGROUND OF THE INVENTION

The following information is provided for the purpose of making knowninformation believed by the applicants to be of possible relevance tothe present invention. No admission is necessarily intended, nor shouldbe construed, that any of the following information constitutes priorart against the present invention.

Antibodies are specific immunoglobulin (Ig) polypeptides produced by thevertebrate immune system in response to challenges by foreign proteins,glycoproteins, cells, or other antigenic foreign substances. The bindingspecificity of such polypeptides to a particular antigen is highlyrefined, with each antibody being almost exclusively directed to theparticular antigen which elicited it.

Two major methods of generating vertebrate antibodies are presentlyutilized: generation insitu by the mammalian B lymphocytes andgeneration in cell culture by B cell hybrids. Antibodies are generatedin situ as a result of the differentiation of immature B lymphocytesinto plasma cells (see Gough (1981), Trends in Biochem Sci, 6:203). Evenwhen only a single antigen is introduced into the immune system of aparticular mammal, a uniform population of antibodies does not result,i.e., the response is polyclonal.

The limited but inherent heterogeneity of polyclonal antibodies isovercome by the use of hybridoma technology to create "monoclonal"antibodies in cell cultures by B cell hybridomas (see Kohler andMilstein (1975), Nature, 256:495-497). In this process, a mammal isinjected with an antigen, and its relatively short-lived, or mortal,splenocytes or lymphocytes are fused with an immortal tumor cell line.The fusion produces hybrid cells or "hybridomas" which are both immortaland capable of producing the genetically-coded antibody of the B cell.

In many applications, the use of monoclonal antibodies produced innon-human animals is severely restricted where the monoclonal antibodiesare to be used in humans. Repeated injections in humans of a "foreign"antibody, such as a mouse antibody, may lead to harmful hypersensitivityreactions, i.e., an anti-idiotypic, or anti-mouse antibody (HAMA),response (see Shawler etal. (1985), Journal of Immunology,135:1530-1535; and Sear et al., J. Biol. Resp. Modifiers, 3:138-150).

Various attempts have already been made to manufacture human-derivedmonoclonal antibodies by using human hybridomas (see Olsson etal.(1980), Proc. Natl. Acad. Sci. USA, 77:5429; and Roder et al. (1986).Methods in Enzymology, 121:140-167). Unfortunately, yields of monoclonalantibodies from human hybridoma cell lines are relatively low comparedto mouse hybridomas. In addition, human cell lines expressingimmunoglobulins are relatively unstable compared to mouse cell lines,and the antibody producing capability of these human cell lines istransient. Thus, while human immunoglobulins are highly desirable, humanhybridoma techniques have not yet reached the stage where humanmonoclonal antibodies with required antigenic specificities can beeasily obtained.

Thus, antibodies of nonhuman origin have been genetically engineered, or"humanized". Humanized antibodies reduce the HAMA response compared tothat expected after injection of a human patient with a mouse antibody.Humanization of antibodies derived from nonhumans, for example, hastaken two principal forms, i.e., chimerization where non-human regionsof immunoglobulin constant sequences are replaced by corresponding humanones (see U.S. Pat. No. 4,816,567 to Cabilly et al., Genentech) andgrafting of complementarity determining regions (CDR) into humanframework regions (FR) (see European Patent Office Application (EPO) 0239 400 to Winter). Some researchers have produced Fv antibodies (seeU.S. Pat. No. 4,642,334 to Moore, DNAX) and single chain Fv (SCFV)antibodies (see U.S. Pat. No. 4,946,778 to Ladner, Genex).

The above patent publications only show the production of antibodyfragments in which some portion of the variable domains is coded for bynonhuman V gene regions. Humanized antibodies to date still retainvarious portions of light and heavy chain variable regions of nonhumanorigin: the chimeric, Fv and single chain Fv antibodies retain theentire variable region of nonhuman origin and CDR-grafted antibodiesretain CDR of nonhuman origin.

Such non-human-derived regions are expected to elicit an immunogenicreaction when administered into a human patient (see Bruggemann et al.(1989), J. Exp. Med., 170:2153-2157; and Lo Buglio (1991), SixthInternational Conference on Monoclonal Antibody Immunoconjugates forCancer, San Diego, Calif.). Thus, it is most desirable to obtain a humanvariable region which is capable of binding to a selected antigen.

One known human carcinoma tumor antigen is tumor-associatedglycoprotein-72 (TAG-72), as defined by monoclonal antibody B72.3 (seeThor et al. (1986) Cancer Res., 46:3118-3124; and Johnson, et al.(1986), Cancer Res., 46:850-857). TAG-72 is associated with the surfaceof certain tumor cells of human origin, specifically the LS174T tumorcell line (American Type Culture Collection (ATCC) No. CL 188), which isa variant of the LS180 (ATCC No. CL 187) colon adeno-carcinoma line.

Numerous murine monoclonal antibodies have been developed which havebinding specificity for TAG-72. Exemplary murine monoclonal antibodiesinclude the "CC" (colon cancer) monoclonal antibodies, which are alibrary of murine monoclonal antibodies developed using TAG-72 purifiedon an immunoaffinity column with an immobilized anti-TAG-72 antibody,B72.3 (ATCC HB-8108) (see EP 394277, to Schlom et al., National CancerInstitute). Certain CC antibodies were deposited with the ATCC: CC49(ATCC No. HB 9459); CC83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9458);CC92 (ATCC No. HB 9454); CC30 (ATCC NO. HB 9457); CC11 (ATCC No. 9455)and CC15 (ATCC No. HB 9460). Various antibodies of the CC series havebeen chimerized (see, for example, EPO 0 365 997 to Mezes et al., TheDow Chemical Company).

It is thus of great interest to develop antibodies against TAG-72containing a light and/or heavy chain variable region(s) derived fromhuman antibodies. However, the prior art simply does not teachrecombinant and immunologic techniques capable of routinely producing ananti-TAG-72 antibody in which the light chain and/or the heavy chainvariable regions have specificity and affinity for TAG-72 and which arederived from human sequences so as to elicit expectedly low or no HAMAresponse. It is known that the function of an immunoglobulin molecule isdependent on its three dimensional structure, which in turn is dependenton its primary amino acid sequence. A change of a few or even one aminoacid can drastically affect the binding function of the antibody, i.e.,the resultant antibodies are generally presumed to be a non-specificimmunoglobulin (NSI), i.e., lacking in antibody character, (see, forexample, U.S. Pat. No. 4,816,567 to Cabilly et al., Genentech).

SUMMARY OF THE INVENTION

Surprisingly, the present invention is capable of meeting many of theseabove-mentioned needs and provides a method for supplying the desiredantibodies. For example, in one aspect, the present invention provides acell capable of expressing a composite antibody having bindingspecificity for TAG-72, said cell being transformed with (a) a DNAsequence encoding at least a portion of a light chain variable region(V_(L)) effectively homologous to the human Subgroup IV germline gene(Hum4 V_(L)); and a DNA sequence segment encoding at least a portion ofa heavy chain variable region (V_(H)) capable of combining with theV_(L) into a three dimensional structure having the ability to bind toTAG-72. As is customary in the art, the term "Hum4 V_(L) " which isitself derived from the designation of a protein--i.e. the variabledomain of the light chain belonging to Subgroup IV of the class of humanκ light chains--can indicate this protein and/or the gene(s) or DNAsequence(s) which encode it.

In one aspect, the present invention concerns a composite antibody orantibody fragment comprising a DNA sequence encoding at least one chainwhich comprises a variable region having a heavy chain (V_(H)) and alight chain (V_(L)), (A) said V_(H) being encoded by a DNA sequencecomprising a subsegment effectively homologous to the V_(H) αTAGgermline gene (V_(H) αTAG), and (B) said V_(L) being encoded by a DNAsequence comprising a subsegment effectively homologous to the humanSubgroup IV germline gene (Hum_(k) IV).

In another aspect, the present invention provides a composite antibodyor antibody fragment having binding specificity for TAG-72, comprising(a) a DNA sequence encoding at least a portion of a light chain variableregion (V_(L)) effectively homologous to the human Subgroup IV germlinegene (Hum4 V_(L)); and a DNA sequence segment encoding at least aportion of a heavy chain variable region (V_(H)) capable of combiningwith the V_(L) into a three dimensional structure having the ability tobind TAG-72.

The invention further includes the aforementioned antibody alone orconjugated to an imaging marker or therapeutic agent. The invention alsoincludes a composition comprising the aforementioned antibody inunconjugated or conjugated form in a pharmaceutically acceptable,non-toxic, sterile carrier.

The invention is also directed to a method for invivo diagnosis ofcancer which comprises administering to an animal containing a tumorexpressing TAG-72 a pharmaceutically effective amount of theaforementioned composition for the insitu detection of carcinomalesions.

The invention is also directed to a method for intraoperative therapywhich comprises (a) administering to a patient containing a tumorexpressing TAG-72 a pharmaceutically effective amount of theaforementioned composition, whereby the tumor is localized, and (b)excising the localized tumors.

Additionally, the invention also concerns a process for preparing andexpressing a composite antibody. Some of these processes are as follows.A process which comprises transforming a cell with a DNA sequenceencoding at least a portion of a light chain variable region (V_(L))effectively homologous to the human Subgroup IV germline gene (Hum4V_(L)); and a DNA sequence segment encoding at least a portion of aheavy chain variable region (V_(H)) which is capable of combining withthe V_(L) to form a three dimensional structure having the ability tobind to TAG-72. A process for preparing a composite antibody or antibodywhich comprises culturing a cell containing a DNA sequence encoding atleast a portion of a light chain variable region (V_(L)) effectivelyhomologous to the human Subgroup IV germline gene (Hum4 V_(L)); and aDNA sequence segment encoding at least a portion of a heavy chainvariable region (V_(H)) capable of combining with the V_(L) into a threedimensional structure having the ability to bind to TAG-72 undersufficient conditions for the cell to express the immunoglobulin lightchain and immunoglobulin heavy chain. A process for preparing anantibody conjugate comprising contacting the aforementioned antibody orantibody with an imaging marker or therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a basic immunoglobulin structure.

FIG. 2, i.e. FIGS. 2A-2G illustrate the nucleotide sequences of V_(H)αTAG, CC46 V_(H), CC49 V_(H), CC83 V_(H) and CC92 V_(H).

FIG. 3, i.e. FIGS. 3A-3E illustrate the amino acid sequences of V_(H)αTAG, CC46 V_(H), CC49 V_(H), CC83 V_(H) and CC92 V_(H).

FIG. 4, i.e. FIGS. 4A-4B illustrate the V_(H) nucleotide and amino acidsequences of antibody B17X2.

FIG. 5, i.e. FIGS. 5A-5B illustrate the mouse germline J-H genes frompNP9.

FIG. 6 illustrates the plasmid map of p49 g1-2.3.

FIG. 7 illustrates the plasmid map of p83 g1-2.3.

FIG. 8, i.e. FIGS. 8A-8B illustrate the entire sequence of HUMVL(+) andHUMVL(-).

FIG. 9 illustrates the human J4 (HJ4) nucleotide sequence and amino acidsequence.

FIG. 10, i.e. FIGS. 10A-10E illustrate the nucleotide sequences, and theamino acid sequences of Hum4 V_(L), ClaI-HindIII segment.

FIG. 11 illustrates a schematic representation of the human germlineSubgroup IV V_(L) gene (Hum4 V_(L)), as the target for the PCR,referring to this gene as the "human germline Subgroup IV gene (HumV_(L))". The 5'-end oligo (HUMV_(L) (+)) and the 3'-end oligo (UUMV_(L)(-)) used to prirne the elongation reactions for Taq polymnerase areshown with half-arrows to indicate the direction of synthesis.

FIG. 12 shows the results of an agarose gel electrophoresis of a PCRreaction to obtain the Hum4 V_(L) gene.

FIG. 13A-13B illustrate the restriction enzyme maps of pRL1000, andprecursor plasmids pSV2neo, pSV2neo-101 and pSV2neo-102. "X" indicateswhere the HindIII site of pSV2neo has been destroyed.

FIG. 14 illustrates a polylinker segment made by synthesizing twooligonucleotides: CH(+) and CH(-). Note that the polylinker could beinserted in both orientations such that the Bam HI site on the left sidecould also be regenerated (and the one on the right side lost).

FIG. 15 illustrates a primer, NE0102SEQ, used for sequencing plasmid DNAfrom several clones of pSV2neo-102. The oligonucleotide is the 21-merhaving the underlined sequence.

FIG. 16 illustrates an autoradiogram depicting the DNA sequence of thepolylinker region both in pSV2neo-102, where the (+) strand of thepolylinker DNA is inserted in the plasmid's sense strand, and inpSV2neo-120, where the (-) strand of the polylinker DNA is inserted inthe plasmid's anti-sense strand.

FIG. 17 illustrates a partial nucleotide sequence segment of pRL1000.

FIG. 18 illustrates the restriction enzyme map of pRL1001.

FIG. 19 illustrates an autoradiogram of DNA sequence for pRL1001 clones.

FIGS. 20A-20C illustrate a competition assay for binding to TAG using acomposite Hum4 V_(L), V_(H) αTAG antibody.

FIG. 21 illustrates a general DNA construction of a single chain,composite Hum4 V_(L), V_(H) αTAG antibody.

FIG. 22, i.e. FIGS. 22A-22C illustrate the nucleotide sequence and aminoacid sequence of SCFV1.

FIGS. 23A-23B shows the construction of plasmid pCGS515/SCFV1.

FIG. 24, i.e. FIGS. 24A-24B show the construction of plasmid pSCFV31.

FIGS. 25A-25B shows the construction of E.coli SCFV expression plasmidscontaining Hum4 V_(L).

FIG. 26, i.e. FIGS. 26A-26D show the DNA sequence and amino acidsequence of Hum4 V_(L) -CC⁴ 9V_(H) SCFV present in pSCFVUHH.

FIG. 27 shows the construction of plasmid pSCFV UHH and a schematic of acombinatorial library of V_(H) genes with Hum4 V_(L).

FIG. 28, i.e. FIGS. 28A-28C illustrate the nucleotide sequence of FLAGpeptide adapter in pATDFLAG.

FIGS. 29A-29B illustrates the construction of pATDFLAG, pHumVL-HumV_(H)(X) and pSC49FLAG.

FIG. 30, i.e. FIGS. 30A-30D illustrate the nucleotide and amino acidsequences of pSC49FLAG.

FIG. 31. i.e. FIGS. 31A-31B show the flow diagram for the discovery ofHum4 V_(L) -V_(H) combinations that compete with prototype TAG-bindingantibodies or mimetics.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic acids, amino acids, peptides, protective groups, active groupsand so on, when abbreviated, are abbreviated according to the IUPAC IUB(Commission on Biological Nomenclature) or the practice in the fieldsconcerned.

The basic immunoglobulin structural unit is set forth in FIG. 1. Theterms "constant" and "variable" are used functionally. The variableregions of both light (V_(L)) and heavy (V_(H)) chains determine bindingrecognition and specificity to the antigen. The constant region domainsof light (C_(L)) and heavy (C_(H)) chains confer important biologicalproperties such as antibody chain association, secretion, transplacentalmobility, complement binding, binding to Fc receptors and the like.

The immunoglobulins of this invention have been developed to address theproblems of the prior art. The methods of this invention produce, andthe invention is directed to, composite antibodies. By "compositeantibodies" is meant immunoglobulins comprising variable regions nothitherto found associated with each other in nature. By "composite Hum4V_(L), V_(H) antibody" means an antibody or immunoreactive fragmentthereof which is characterized by having at least a portion of the V_(L)region encoded by DNA derived from the Hum4 V_(L) germline gene and atleast a portion of a V_(H) region capable of combining with the V_(L) toform a three dimensional structure having the ability to bind to TAG-72.

The composite Hum4 V_(L), V_(H) antibodies of the present inventionassume a conformation having an antigen binding site which bindsspecifically and with sufficient strength to TAG-72 to form a complexcapable of being isolated by using standard assay techniques (e.g.,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like). Preferably, the composite Hum4 V_(L), V_(H) antibodies ofthe present invention have an antigen binding affinity or aviditygreater than 10⁵ M⁻¹, more preferably greater than 10⁶ M⁻¹ and mostpreferably greater than 10⁸ M⁻¹. For a discussion of the techniques forgenerating and reviewing immunoglobulin binding affinities see Munson(1983), Methods Enzymol., 92:543-577 and Scatchard (1949), Ann. N.Y.Acad. Sci., 51:660-672.

Human antibody kappa chains have been classified into four subgroups onthe basis of invariant amino acid sequences (see, for example, Kabat etal. (1991), Sequences of Proteins of Immunological Interest (4th ed.),published by The U.S. Department of Health and Human Services). Thereappear to be approximately 80 human V_(K) genes, but only one SubgroupIV V_(K) gene has been identified in the human genome (see Klobeck, etal. (1985), Nucleic Acids Research, 13:6516-6528). The nucleotidesequence of Hum4 V_(L) is set forth in Kabat et al. (1991), supra.

It has been found, quite surprisingly, that an immunoglobulin having alight chain with at least a portion of the V_(L) encoded by a genederived from Hum4 V_(L) may, if combined with a suitable V_(H), havebinding specificity for TAG-72.

The type of J_(L) gene segment selected is not critical to theinvention, in that it is expected that any J_(L), if present, canassociate with the Hum4 V_(L). The present invention obviouslycontemplates the Hum4 V_(L) in association with a human J_(K) sequence.The five human J_(K) sequences are set forth in Heiter et al. (1982),The Journal of Biological Chemistry, 357:1516-1522. However, the presentinvention is not intended to be limited to the human J_(K). The presentinvention specifically contemplates the Hum4 V_(L) in association withany of the at least six human J.sub.λ genes (see Hollis et al. (1982),Nature, 296:321-325).

An exemplary technique for engineering the Hum4 V_(L) with selectedJ_(L) segments includes synthesizing a primer having a so-called"wagging tail", that does not hybridize with the target DNA; thereafter,the sequences are amplified and spliced together by overlap extension(see Horton et al. (1989), Gene, 77:61-68).

The C_(L) of the composite Hum4 V_(L), V_(H) antibodies is not criticalto the invention. To date, the Hum4 V_(L) has only been reported ashaving been naturally rearranged with the single C_(K) gene (see Heiteret al. (1980), Cell, 22:197-207). However, the present invention is notintended to be limited to the C_(K) light chain constant domain. Thatis, the C_(L) gene segment may also be any of the at least six C.sub.λgenes (see Hollis et al., supra).

The DNA encoding the heavy chain variable region consists roughly of aheavy chain variable (V_(H)) gene sequence, a heavy chain diversity(D_(H)) gene sequence, and a heavy chain joining (J_(H)) gene sequence.

The present invention is directed to any V_(H) capable of combining witha light chain variable region effectively homologous to the light chainvariable region encoded by the human Subgroup IV germline gene, to forma three dimensional structure having the ability to bind to TAG-72.

The choice of D_(H) and J_(H) segment of the composite Hum4 V_(L), V_(H)antibody is not critical to the present invention. Obviously, human andmurine D_(H) and J_(H) gene segments are contemplated, provided that agiven combination does not significantly decrease binding to TAG-72.Specifically, when utilizing CC46 V_(H), CC49 V_(H), CC83 V_(H) and CC92V_(H), the composite Hum4 V_(L), V_(H) antibody will be designed toutilize the D_(H) and J_(H) segments which naturally associated withthose V_(H) of the respective hybridomas (see FIGS. 2 and 3). Exemplarymurine and human D_(H) and J_(H) sequences are set forth in Kabat et al.(1991), supra. An exemplary technique for engineering such selectedD_(H) and J_(H) segments with a V_(H) sequence of choice includessynthesizing selected oligonucleotides, annealing and ligating in acloning procedure (see, Horton et al., supra).

In a specific embodiment the composite Hum4 V_(L), V_(H) antibody willbe a "composite Hum4 V_(L), V_(H) αTAG antibody", means an antibody orimmunoreactive fragment thereof which is characterized by having atleast a portion of the V_(L) region encoded by DNA derived from the Hum4V_(L) germline gene and at least a portion of the V_(H) region encodedby DNA derived from the V_(H) αTAG germline gene, which is known in theart (see, for example, EPO 0 365 997 to Mezes et al., The Dow ChemicalCompany). FIG. 2 shows the nucleotide sequence Of V_(H) αTAG, and thenucleotide sequences encoding the V_(H) Of the CC46, CC49, CC83 and CC92antibodies, respectively. FIG. 3 shows the corresponding amino acidsequences of V_(H) αTAG, CC46 V_(H), CC49 V_(H), CC83 V_(H) and CC92V_(H).

A comparison of the nucleotide and amino acid sequences of V_(H) αTAG,CC46 V_(H), CC49 V_(H), CC83 V_(H) and CC92 V_(H) shows that those CCantibodies are derived from V_(H) αTAG. Somatic mutations occurringduring productive rearrangement of the V_(H) derived from V_(H) αTAG ina B cell gave rise to some nucleotide changes that may or may not resultin a homologous amino acid change between the productively rearrangedhybridomas (see, EPO 0 365 997).

Because the nucleotide sequences of the V_(H) αTAG and Hum4 V_(L)germline genes have been provided herein, the present invention isintended to include other antibody genes which are productivelyrearranged from the V_(H) αTAG germline gene. Other antibodies encodedby DNA derived from V_(H) αTAG may be identified by using ahybridization probe made from the DNA or RNA of the V_(H) αTAG orrearranged genes containing the recombined V_(H) αTAG. Specifically, theprobe will include of all or a part of the V_(H) αTAG germline gene andits flanking regions. By "flanking regions" is meant to include thoseDNA sequences from the 5' end of the V_(H) αTAG to the 3' end of theupstream gene, and from 3' end of the V_(H) αTAG to the 5' end of thedownstream gene.

The CDR from the variable region of antibodies derived from V_(H) αTAGmay be grafted onto the FR of selected V_(H), i.e., FR of a humanantibody (see EPO 0 239 400 to Winter). For example, the cell line,B17X2, expresses an antibody utilizing a variable light chain encoded bya gene derived from Hum4 V_(L) and a variable heavy chain which makes astable V_(L) and V_(H) combination (see Marsh et al. (1985), NucleicAcids Research, 13:6531-6544; and Polke et al. (1982), Immunobiol.163:95-109. The nucleotide sequence of the V_(H) chain for B17X2 isshown in FIG. 4. The B17X2 cell line is publicly available from Dr.Christine Polke, Universitats-Kinderklinik, Josef-Schneider-Str. 2, 8700Wurzburg, FRG). B17X2 is directed to N-Acetyl-D-Glucosamine and is notspecific for TAG-72.

However, consensus sequences of antibody derived from the CDR1 of V_(H)αTAG (amino acid residues 31 to 35 of FIG. 3) may be inserted into B17X2(amino acid residues 31 to 37 of FIG. 4) and the CDR2 of V_(H) αTAG(amino residues 50 to 65 of FIG. 3) may be inserted into B17X2 (aminoacid residues 52 to 67 of FIG. 4). The CDR3 may be replaced by any D_(H)and J_(H) sequence which does not affect the binding of the antibody forTAG-72 but, specifically, may be replaced by the CDR3 of an antibodyhaving its V_(H) derived from V_(H) αTAG, e.g., CC46, CC49, CC83 andCC92. Exemplary techniques for such replacement are set forth in Hortonet al., supra.

The C_(H) domains of immunoglobulin heavy chain derived from V_(H) αTAGgenes, for example, may be changed to a human sequence by knowntechniques (see, U.S. Pat. No. 4,816,567 to Cabilly, Genentech). C_(H)domains may be of various complete or shortened human isotypes, i.e.,IgG (e.g., IgG₁, IgG₂, IgG₃, and IgG₄), IgA (e.g., IgA1 and IgA2), IgD,IgE, IgM, as well as the various allotypes of the individual groups (seeKabat et al. (1991), supra).

Given the teachings of the present invention, it should be apparent tothe skilled artisan that human V_(H) genes can be tested for theirability to produce an anti-TAG-72 immunoglobulin combination with theHum4 V_(L) gene. The V_(L) may be used to isolate a gene encoding for aV_(H) having the ability to bind to TAG-72 to test myriad combinationsof Hum4 V_(L) and V_(H) that may not naturally occur in nature, e.g., bygenerating a combinatorial library using the Hum4 V_(L) gene to select asuitable V_(H). Examples of these enabling technologies includescreening of combinatorial libraries of V_(L) -V_(H) combinations usingan Fab or single chain antibody (SCFV) format expressed on the surfacesof fd phage (Clackson, et al. (1991), Nature, 352:624-628), or using a Aphage system for expression of Fv's or Fabs (Huse, et al. (1989),Science, 246:1275-1281). However, according to the teachings set forthherein, it is now possible to clone SCFV antibodies in E.coli, andexpress the SCFVs as secreted soluble proteins. SCFV proteins producedin E. coli that contain a Hum4 V_(L) gene can be screened for binding toTAG-72 using, for example, a two-membrane filter screening system(Skerra, et al. (1991), Analytical Biochemistry, 196:151-155).

The desired gene repertoire can be isolated from human genetic materialobtained from any suitable source, e.g., peripheral blood lymphocytes,spleen cells and lymph nodes of a patient with tumor expressing TAG-72.In some cases, it is desirable to bias the repertoire for a preselectedactivity, such as by using as a source of nucleic acid, cells (sourcecells) from vertebrates in any one of various stages of age, health andimmune response.

Cells coding for the desired sequence may be isolated, and genomic DNAfragmented by one or more restriction enzymes. Tissue (e.g., primary andsecondary lymph organs, neoplastic tissue, white blood cells fromperipheral blood and hybridomas) from an animal exposed to TAG-72 may beprobed for selected antibody producing B cells. Variability among Bcells derived from a common germline gene may result from somaticmutations occurring during productive rearrangement.

Generally, a probe made from the genomic DNA of a germline gene orrearranged gene can be used by those skilled in the art to findhomologous sequences from unknown cells. For example, sequenceinformation obtained from Hum4 V_(L) and V_(H) αTAG may be used togenerate hybridization probes for naturally-occurring rearranged Vregions, including the 5' and 3' nontranslated flanking regions. Thegenomic DNA may include naturally-occurring introns for portionsthereof, provided that functional splice donor and splice acceptorregions had been present in the case of mammalian cell sources.

Additionally, the DNA may also be obtained from a cDNA library. mRNAcoding for heavy or light chain variable domain may be isolated from asuitable source, either mature B cells or a hybridoma culture, employingstandard techniques of RNA isolation. The DNA or amino acids also may besynthetically synthesized and constructed by standard techniques ofannealing and ligating fragments (see Jones, et al. (1986), Nature,321:522-525; Reichmann et al., (1988), Nature, 332:323-327; Sambrook etal. (1989), supra and Merrifield et al. (1963), J. Amer. Chem. Soc.,85:2149-2154). Heavy and light chains may be combined invitro to gainantibody activity (see Edelman, et al. (1963), Proc. Natl. Acad. Sci.USA, 50:753).

The present invention also contemplates a gene library of V_(H) αTAGhomologs, preferably human homologs of V_(H) αTAG. By "homolog" is meanta gene coding for a V_(H) region (not necessarily derived from, or eveneffectively homologous to, the V_(H) αTAG germline gene) capable ofcombining with a light chain variable region effectively homologous tothe light chain variable region encoded by the human Subgroup IVgermline gene, to form a three dimensional structure having the abilityto bind to TAG-72.

Preferably, the gene library is produced by a primer extension reactionor combination of primer extension reactions as described herein. TheV_(H) αTAG homologs are preferably in an isolated form, i.e.,substantially free of materials such as, for example, primer extensionreaction agents and/or substrates, genomic DNA segments, and the like.The present invention thus is directed to cloning the V_(H) αTAG-codingDNA homologs from a repertoire comprised of polynucleotide codingstrands, such as genomic material containing the gene expressing thevariable region or the messenger RNA (mRNA) which represents atranscript of the variable region. Nucleic acids coding for V_(H)αTAG-coding homologs can be derived from cells producing IgA, IgD, IgE,IgG or IgM, most preferably from IgM and IgG, producing cells.

The V_(H) αTAG-coding DNA homologs may be produced by primer extension.The term "primer" as used herein refers to a polynucleotide whetherpurified from a nucleic acid restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complimentary to a nucleic acid strand isinduced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase, reverse transcriptase and thelike, and at a suitable temperature and pH.

Preferably, the V_(H) αTAG-coding DNA homologs may be produced bypolymerase chain reaction (PCR) amplification of double stranded genomicor cDNA, wherein two primers are used for each coding strand of nucleicacid to be exponentially amplified. The first primer becomes part of thenonsense (minus or complementary) strand and hybridizes to a nucleotidesequence conserved among V_(H) (plus) strands within the repertoire. PCRis described in Mullis et al. (1987), Meth. Enz., 155:335-350; and PCRTechnology, Erlich (ed.) (1989). PCR amplification of the mRNA fromantibody-producing cells is set forth in Orlandi et al. (1989), Proc.Natl. Acad. Sci., USA, 86:3387-3837.

According to a preferred method, the V_(H) αTAG-coding DNA homologs areconnected via linker to form a SCFV having a three dimensional structurecapable of binding TAG-72. The SCFV construct can be in a V_(L) -L-V_(H)or V_(H) -L-V_(L) configuration. For a discussion of SCFV see Bird etal. (1988), Science, 242:423-426. The design of suitable peptide linkerregions is described in U.S. Pat. No. 4,704,692 to Ladner et al., Genex.

The nucleotide sequence of a primer is selected to hybridize with aplurality of immunoglobulin heavy chain genes at a site substantiallyadjacent to the V_(H) αTAG-coding DNA homolog so that a nucleotidesequence coding for a functional (capable of binding) polypeptide isobtained. The choice of a primer's nucleotide sequence depends onfactors such as the distance on the nucleic acid from the region codingfor the desired receptor, its hybridization site on the nucleic acidrelative to any second primer to be used, the number of genes in therepertoire it is to hybridize to, and the like. To hybridize to aplurality of different nucleic acid strands of V_(H) αTAG-coding DNAhomolog, the primer must be a substantial complement of a nucleotidesequence conserved among the different strands.

The peptide linker may be coded for by the nucleic acid sequences thatare part of the polynucleotide primers used to prepare the various genelibraries. The nucleic acid sequence coding for the peptide linker canbe made up of nucleic acids attached to one of the primers or thenucleic acid sequence coding for the peptide linker may be derived fromnucleic acid sequences that are attached to several polynucleotideprimers used to create the gene libraries. Additionally,noncomplementary bases or longer sequences can be interspersed into theprimer, provided the primer sequence has sufficient complementarily withthe sequence of the strand to be synthesized or amplified tonon-randomly hybridize therewith and thereby form an extension productunder polynucleotide synthesizing conditions (see Horton et al. (1989),Gene, 77:61-68).

Exemplary human V_(H) sequences from which complementary primers may besynthesized are set forth in Kabat et al. (1991), supra; Humphries etal. (1988), Nature, 331:446-449; Schroeder et al. (1990), Proc. Natl.Acad. Sci. USA, 87:6146-6150; Berman et al. (1988), EMBO Journal,7:727-738; Lee et al. (1987), J. Mol. Biol., 195:761-768); Marks et al.(1991), Eur. J. Immunol., 21:985-991; Willems, et al. (1991), J.Immunol., 146:3646-3651; and Person et al. (1991), Proc. Natl. Acad.Sci. USA, 88:2432-2436. To produce V_(H) coding DNA homologs, firstprimers are therefore chosen to hybridize to (i.e. be complementary to)conserved regions within the J region, CH1 region, hinge region, CH2region, or CH3 region of immunoglobulin genes and the like. Secondprimers are therefore chosen to hydribidize with a conserved nucleotidesequence at the 5' end of the V_(H) αTAG-coding DNA homolog such as inthat area coding for the leader or first framework region.

Alternatively, the nucleic acid sequences coding for the peptide linkermay be designed as part of a suitable vector. As used herein, the term"expression vector" refers to a nucleic acid molecule capable ofdirecting the expression of genes to which they are operatively linked.The choice of vector to which a V_(H) αTAG-coding DNA homologs isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., replication or protein expression,and the host cell (either prokaryotic or eukaryotic) to be transformed,these being limitations inherent in the art of constructing recombinantDNA molecules. In preferred embodiments, the eukaryotic cell expressionvectors used include a selection marker that is effective in aneukaryotic cell, preferably a drug resistant selection marker.

Expression vectors compatible with prokaryotic cells are well known inthe art and are available from several commercial sources. Typical ofvector plasmids suitable for prokaryotic cells are pUC8, pUC9, pBR322,and pBR329 available from BioRad Laboratories, (Richmond, Calif.), andpPL and pKK223 available from Pharmacia, (Piscataway, N.J.).

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAhomolog. Typical of vector plasmids suitable for eukaryotic cells arepSV2neo and pSV2gpt (ATCC), pSV_(L) and pKSV-10 (Pharmacia),pBPV-1/PML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC).

The use of viral expression vectors to express the genes of the V_(H)αTAG-coding DNA homologs is also contemplated. As used herein, the term"viral expression vector" refers to a DNA molecule that includes apromoter sequence derived from the long terminal repeat (LTR) region ofa viral genome. Exemplary phage include λ phage and fd phage (see,Sambrook, et al. (1989), Molecular Cloning: A Laboratory Manual, (2nded.), and McCafferty et al. (1990), Nature, 6301:552-554.

The population of V_(H) αTAG-coding DNA homologs and vectors are thencleaved with an endonuclease at shared restriction sites. A variety ofmethods have been developed to operatively link DNA to vectors viacomplementary cohesive termini. For instance, complementary cohesivetermini can be engineered into the V_(H) αTAG-coding DNA homologs duringthe primer extension reaction by use of an appropriately designedpolynucleotide synthesis primer, as previously discussed. Thecomplementary cohesive termini of the vector and the DNA homolog arethen operatively linked (ligated) to produce a unitary double strandedDNA molecule.

The restriction fragments of Hum4 V_(L) -coding DNA and the V_(H)αTAG-coding DNA homologs population are randomly ligated to the cleavedvector. A diverse, random population is produced with each vector havinga V_(H) αTAG-coding DNA homolog and Hum4 V_(L) -coding DNA located inthe same reading frame and under the control of the vector's promoter.

The resulting single chain construct is then introduced into anappropriate host to provide amplification and/or expression of acomposite Hum4 V_(L), V_(H) αTAG homolog single chain antibody.Transformation of appropriate cell hosts with a recombinant DNA moleculeof the present invention is accomplished by methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (1972),Proceedings National Academy of Science, USA, 69:2110; and Sambrook, etal. (1989), supra. With regard to the transformation of vertebrate cellswith retroviral vectors containing rDNAs, see, for example, Sorge et al.(1984), Mol. Cell. Biol., 4:1730-1737; Graham et al. (1973), Virol.,52:456; and Wigler et al. (1979), Proceedings National Academy ofSciences, USA, 76:1373-1376.

Exemplary prokaryotic strains that may be used as hosts include E.coli,Bacilli, and other enterobacteriaceae such as Salmonella typhimurium,and various Pseudomonas. Common eukaryotic microbes include S.cerevisiae and Pichia pastoris. Common higher eukaryotic host cellsinclude Sp2/0, VERO and HeLa cells, Chinese hamster ovary (CHO) celllines, and W138, BHK, COS-7 and MDCK cell lines. Furthermore, it is nowalso evident that any cell line producing Hum4 V_(L), e.g., the B17X2human cell line, can be used as a recipient human cell line forintroduction of a V_(H) gene complementary to the Hum4 V_(L) whichallows binding to TAG-72. For example, the B17X2 heavy chain may begenetically modified to not produce the endogenous heavy chain by wellknown methods; in this way, glycosylation patterns of the antibodyproduced would be human and not non-human derived.

Successfully transformed cells, i.e., cells containing a gene encoding acomposite Hum4 V_(L), V_(H) αTAG homolog single chain antibodyoperatively linked to a vector, can be identified by any suitable wellknown technique for detecting the binding of a receptor to a ligand.Preferred screening assays are those where the binding of the compositeHum4 V_(L), V_(H) αTAG homolog single chain antibody to TAG-72 producesa detectable signal, either directly or indirectly. Screening forproductive Hum4 V_(L) and V_(H) αTAG homolog combinations, or in otherwords, testing for effective antigen binding sites to TAG-72 is possibleby using, for example, a radiolabeled or biotinylated screening agent,e.g., antigens, antibodies (e.g., B72.3, CC49, CC83, CC46, CC92, CC30,CC11 and CC15) or anti-idiotypic antibodies (see Huse et al., supra, andSambrook et al., supra); or the use of marker peptides to the NH₂ - orCOOH-terminus of the SCFV construct (see Hopp et al. (1988),Biotechnology, 6:1204-1210). Of course, the Hum4 V_(L) -coding DNA andthe V_(H) αTAG-coding DNA homologs may be expressed as individualpolypeptide chains (e.g., Fv) or with whole or fragmented constantregions (e.g., Fab, and F(ab')₂). Accordingly, the Hum4 V_(L) -codingDNA and the V_(H) αTAG-coding DNA homologs may be individually insertedinto a vector containing a C_(L) or C_(H) or fragment thereof,respectively. For a teaching of how to prepare suitable vectors see EPO0 365 997 to Mezes et al., The Dow Chemical Company.

DNA sequences encoding the light chain and heavy chain of the compositeHum4 V_(L), V_(H) antibody may be inserted into separate expressionvehicles, or into the same expression vehicle. When coexpressed withinthe same organism, either on the same or the different vectors, afunctionally active Fv is produced. When the V_(H) αTAG-coding DNAhomolog and Hum4 V_(L) polypeptides are expressed in differentorganisms, the respective polypeptides are isolated and then combined inan appropriate medium to form a Fv. See Greene et al., Methods inMolecular Biology, Vol. 9, Wickner et al. (ed.); and Sambrook et al.,supra).

Subsequent recombinations can be effected through cleavage and removalof the Hum4 V_(L) -coding DNA sequence to use the V_(H) αTAG-coding DNAhomologs to produce Hum4 V_(L) -coding DNA homologs. To produce a Hum4V_(L) -coding DNA homolog, first primers are chosen to hybridize with(i.e. be complementary to) a conserved region within the J region orconstant region of immunoglobulin light chain genes and the like. Secondprimers become part of the coding (plus) strand and hybridize to anucleotide sequence conserved among minus strands. Hum4 V_(L) -codingDNA homologs are ligated into the vector containing the V_(H)αTAG-coding DNA homolog, thereby creating a second population ofexpression vectors. The present invention thus is directed to cloningthe Hum4 V_(L) -coding DNA homologs from a repertoire comprised ofpolynucleotide coding strands, such as genomic material containing thegene expressing the variable region or the messenger RNA (mRNA) whichrepresents a transcript of the variable region. It is thus possible touse an iterative process to define yet further, composite antibodies,using later generation V_(H) αTAG-coding DNA homologs and Hum4 V_(L)-coding DNA homologs.

The present invention further contemplates genetically modifying theantibody variable and constant regions to include effectively homologousvariable region and constant region amino acid sequences. Generally,changes in the variable region will be made in order to improve orotherwise modify antigen binding properties of the receptor. Changes inthe constant region of the antibody will, in general, be made in orderto improve or otherwise modify biological properties, such as complementfixation, interaction with membranes, and other effector functions.

"Effectively homologous" refers to the concept that differences in theprimary structure of the variable region may not alter the bindingcharacteristics of the antibody. Normally, a DNA sequence is effectivelyhomologous to a second DNA sequence if at least 70 percent, preferablyat least 80 percent, and most preferably at least 90 percent of theactive portions of the DNA sequence are homologous. Such changes arepermissible in effectively homologous amino acid sequences so long asthe resultant antibody retains its desired property.

If there is only a conservative difference between homologous positionsof sequences, they can be regarded as equivalents under certaincircumstances. General categories of potentially equivalent amino acidsare set forth below, wherein amino acids within a group may besubstituted for other amino acids in that group: (1) glutamic acid andaspartic acid; (2) hydrophobic amino acids such as alanine, valine,leucine and isoleucine; (3) asparagine and glutamine; (4) lysine andarginine and (5) threonine and serine.

Exemplary techniques for nucleotide replacement include the addition,deletion or substitution of various nucleotides, provided that theproper reading frame is maintained. Exemplary techniques include usingpolynucleotide-mediated, site-directed mutagenesis, i.e., using a singlestrand as a template for extension of the oligonucleotide to produce astrand containing the mutation (see Zoller et al. (1982), Nuc. AcidsRes., 10:6487-6500; Norris et al. (1983), Nuc. Acids Res., 11:5103-5112;Zoller et al. (1984), DNA, 3:479-488; and Kramer et al. (1982), Nuc.Acids Res., 10:6475-6485) and polymerase chain reaction exponentiallyamplifying DNA invitro using sequence specified oligonucleotides toincorporate selected changes (see PCR Technology: Principles andApplications for DNA Amplification, Erlich, (ed.) (1989); and Horton etal., supra).

Further, the antibodies may have their constant region domain modified,i.e., the C_(L), CH₁, hinge, CH₂, CH₃ and/or CH₄ domains of an antibodypolypeptide chain may be deleted, inserted or changed (see EPO 327 378A1 to Morrison et al., the Trustees of Columbia University; U.S. Pat.No. 4,642,334 to Moore et al., DNAX; and U.S. Pat. No. 4,704,692 toLadner et al., Genex).

Once a final construct is obtained, the composite Hum4 V_(L), V_(H)antibodies may be produced in large quantities by injecting the hostcell into the peritoneal cavity of pristane-primed mice, and after anappropriate time (about 1-2 weeks), harvesting ascites fluid from themice, which yields a very high titer of homogeneous composite Hum4V_(L), V_(H) antibodies, and isolating the composite Hum4 V_(L), V_(H)antibodies by methods well known in the art (see Stramignoni et al.(1983), Intl. J. Cancer, 31:543-552). The host cell is grown invivo, astumors in animals, the serum or ascites fluid of which can provide up toabout 50 mg/mL of composite Hum4 V_(L), V_(H) antibodies. Usually,injection (preferably intraperitoneal) of about 10⁶ to 10⁷histocompatible host cells into mice or rats will result in tumorformation after a few weeks. It is possible to obtain the composite Hum4V_(L), V_(H) antibodies from a fermentation culture broth of prokaryoticand eukaryotic cells, or from inclusion bodies of E.coli cells (seeBuckholz and Gleeson (1991), BIO/TECHNOLOGY, 9:1067-1072. The compositeHum4 V_(L), V_(H) antibodies can then be collected and processed bywell-known methods (see generally, Immunological Methods, vols. I & II,eds. Lefkovits, I. and Pernis, B., (1979 & 1981) Academic Press, NewYork, N.Y.; and Handbook of Experimental Immunology, ed. Weir, D.,(1978) Blackwell Scientific Publications, St. Louis, Mo.).

The composite Hum4 V_(L), V_(H) antibodies can then be stored in variousbuffer solutions such as phosphate buffered saline (PBS), which gives agenerally stable antibody solution for further use.

Uses

The composite Hum4 V_(L), V_(H) antibodies provide unique benefits foruse in a variety of cancer treatments. In addition to the ability tobind specifically to malignant cells and to localize tumors and not bindto normal cells such as fibroblasts, endothelial cells, or epithelialcells in the major organs, the composite Hum4 V_(L), V_(H) antibodiesmay be used to greatly minimize or eliminate HAMA responses thereto.Moreover, TAG-72 contains a variety of epitopes and thus it may bedesirable to administer several different composite Hum4 V_(L), V_(H)antibodies which utilize a variety of V_(H) in combination with Hum4V_(L).

Specifically, the composite Hum4 V_(L), V_(H) antibodies are useful for,but not limited to, invivo and invitro uses in diagnostics, therapy,imaging and biosensors.

The composite Hum4 V_(L), V_(H) antibodies may be incorporated into apharmaceutically acceptable, non-toxic, sterile carrier. Injectablecompositions of the present invention may be either in suspension orsolution form. In solution form the complex (or when desired theseparate components) is dissolved in a pharmaceutically acceptablecarrier. Such carriers comprise a suitable solvent, preservatives suchas benzyl alcohol, if needed, and buffers. Useful solvents include, forexample, water, aqueous alcohols, glycols, and phosphonate or carbonateesters. Such aqueous solutions generally contain no more than 50 percentof the organic solvent by volume.

Injectable suspensions require a liquid suspending medium, with orwithout adjuvants, as a carrier. The suspending medium can be, forexample, aqueous polyvinyl-pyrrolidone, inert oils such as vegetableoils or highly refined mineral oils, or aqueous carboxymethylcellulose.Suitable physiologically-acceptable adjuvants, if necessary to keep thecomplex in suspension, may be chosen from among thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and the alginates.Many surfactants are also useful as suspending agents, for example,lecithin, alkylphenol, polyethylene oxide adducts,naphthalenesulfonates, alkylbenzenesulfonates, and the polyoxyethylenesorbitan esters. Many substances which effect the hydrophobicity,density, and surface tension of the liquid suspension medium can assistin making injectable suspensions in individual cases. For example,silicone antifoams, sorbitol, and sugars are all useful suspendingagents.

Methods of preparing and administering conjugates of the composite Hum4V_(L), V_(H) antibody, and a therapeutic agent are well known or readilydetermined. Moreover, suitable dosages will depend on the age and weightof the patient and the therapeutic agent employed and are well known orreadily determined.

Conjugates of a composite Hum4 V_(L), V_(H) antibody and an imagingmarker may be administered in a pharmaceutically effective amount forthe invivo diagnostic assays of human carcinomas, or metastases thereof,in a patient having a tumor that expresses TAG-72 and then detecting thepresence of the imaging marker by appropriate detection means.

Administration and detection of the conjugates of the composite Hum4V_(L), V_(H) antibody and an imaging marker, as well as methods ofconjugating the composite Hum4 V_(L), V_(H) antibody to the imagingmarker are accomplished by methods readily known or readily determined.The dosage of such conjugate will vary depending upon the age and weightof the patient. Generally, the dosage should be effective to visualizeor detect tumor sites, distinct from normal tissues. Preferably, aone-time dosage will be between 0.1 mg to 200 mg of the conjugate of thecomposite Hum4 V_(L), V_(H) antibody and imaging marker per patient.

Examples of imaging markers which can be conjugated to the compositeHum4 V_(L), V_(H) antibody are well known and include substances whichcan be detected by diagnostic imaging using a gamma scanner or hand heldgamma probe, and substances which can be detected by nuclear magneticresonance imaging using a nuclear magnetic resonance spectrometer.

Suitable, but not limiting, examples of substances which can be detectedusing a gamma scanner include ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹¹¹ In, ¹⁰⁵ Rh, ¹⁵³Sm, ⁶⁷ Cu, ⁶⁷ Ga, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re and ^(99m) Tc. Anexample of a substance which can be detected using a nuclear magneticresonance spectrometer is gadolinium.

Conjugates of a composite Hum4 V_(L), V_(H) antibodies and a therapeuticagent may be administered in a pharmaceutically effective amount for theinvivo treatment of human carcinomas, or metastases thereof, in apatient having a tumor that expresses TAG-72. A "pharmaceuticallyeffective amount" of the composite Hum4 V_(L) antibody means the amountof said antibody (whether unconjugated, i.e., a naked antibody, orconjugated to a therapeutic agent) in the pharmaceutical compositionshould be sufficient to achieve effective binding to TAG-72.

Exemplary naked antibody therapy includes, for example, administeringheterobifunctional composite Hum4 V_(L), V_(H) antibodies coupled orcombined with another antibody so that the complex binds both to thecarcinoma and effector cells, e.g., killer cells such as T cells, ormonocytes. In this method, the composite Hum4 V_(L) antibody-therapeuticagent conjugate can be delivered to the carcinoma site thereby directlyexposing the carcinoma tissue to the therapeutic agent. Alternatively,naked antibody therapy is possible in which antibody dependent cellularcytoxicity or complement dependent cytotoxicity is mediated by thecomposite Hum4 V_(L) antibody.

Examples of the antibody-therapeutic agent conjugates which can be usedin therapy include antibodies coupled to radionuclides, such as ¹³¹ I,⁹⁰ Y, ¹⁰⁵ Rh, ⁴⁷ Sc, ⁶⁷ Cu, ²¹² Bi, ²¹¹ At, ⁶⁷ Ga, ¹²⁵ I, ¹⁸⁶ Re, ¹⁸⁸Re, ¹⁷⁷ Lu, ^(99m) Tc, ¹⁵³ Sm, ¹²³ I and ¹¹¹ In; to drugs, such asmethotrexate, adriamycin; to biological response modifiers, such asinterferon and to toxins, such as ricin.

Methods of preparing and administering conjugates of the composite Hum4V_(L), V_(H) antibodies and a therapeutic agent are well known orreadily determined. The pharmaceutical composition may be administeredin a single dosage or multiple dosage form. Moreover, suitable dosageswill depend on the age and weight of the patient and the therapeuticagent employed and are well known or readily determined.

Composite Hum4 V_(L), V_(H) antibodies, and particularly composite Hum4V_(L), V_(H) single chain antibodies thereof, are particularly suitablefor radioimmunoguided surgery (RIGS). In RIGS, an antibody labeled withan imaging marker is injected into a patient having a tumor thatexpresses TAG-72. The antibody localizes to the tumor and is detected bya hand-held gamma detecting probe (GDP). The tumor is then excised (seeMartin et al. (1988), Amer. J. Surg., 156:386-392; and Martin et al.(1986), Hybridoma, 5:S97-S108). An exemplary GDP is the Neoprobe™scanner, commercially available from Neoprobe Corporation, Columbus,Ohio. The relatively small size and human character of the compositeHum4 V_(L), V_(H) single chain antibodies will accelerate whole bodyclearance and thus reduce the waiting period after injection beforesurgery can be effectively initiated.

Administration and detection of the composite Hum4 V_(L), V_(H)antibody-imaging marker conjugate may be accomplished by methods wellknown or readily determined.

The dosage will vary depending upon the age and weight of the patient,but generally a one-time dosage of 0.1 mg to 200 mg of the compositeHum4 V_(L), V_(H) antibody-marker conjugate per patient is administered.

EXAMPLES

The following non-limiting examples are merely for illustration of theconstruction and expression of composite Hum4 V_(L), V_(H) antibodies.All temperatures not otherwise indicated are Centigrade. All percentsnot otherwise indicated are by weight.

Example 1

CC49 and CC83 were isolated from their respective hybridomas using pNP9as a probe (see FIG. 5). CC49 V_(H) was obtained from p49 g1-2.3 (seeFIG. 6) and CC83 V_(H) was obtained from p83 g1-2.3 (see FIG. 7),following the procedures set forth in EPO 0 365 997.

DNA encoding an antibody light chain was isolated from a sample of bloodfrom a human following the protocol of Madisen et. al. (1987), Am. J.Med. Genet., 27:379-390), with several modifications. Two 5 mLpurple-cap Vacutainer tubes (containing EDTA as an anticoagulant) werefilled with blood and stored at ambient temperature for 2 hours. Thesamples were transferred to two 4.5 mL centrifuge tubes. To each tubewas added 22.5 mL of filter-sterilized erythrocycte lysate buffer (0.155M NH₄ Cl and 0.17 M Tris, pH 7.65, in a volume ratio of 9:1), andincubated at 37° C. for 6.5 minutes. The tubes became dark red due tothe lysed red blood cells. The samples were centrifuged at 9° C. for 10minutes, using an SS-34 rotor and a Sorvall centrifuge at 5,300revolutions per minute (rpm) (˜3,400×g). The resulting white cellpellets were resuspended in 25 mL of 0.15 M NaCl solution. The whiteblood cells were then centrifuged as before. The pellets wereresuspended in 500 μL of 0.15 M NaCl and transferred to 1.5 mLmicrocentrifuge tubes. The cells were pelleted again for 3 minutes, thistime in the microcentrifuge at 3,000 rpm. Very few red blood cellsremained on the pellet. After the supernatants were decanted from the 2microcentrifuge tubes, 0.6 mL high TE buffer (100 mM Tris, pH 8.0) wasadded. The tubes were hand-shaken for between 10 and 15 minutes. Theresulting viscous solution was extracted with phenol, phenol-chloroformand finally with just chloroform as described in Sambrook et al., supra.To 3.9 mL of pooled extracted DNA solution were added 0.4 mL NaOAc (3 M,pH 5), and 10 mL 100 percent ethanol. A white stringy precipitate wasrecovered with a yellow pipette tip, transferred into a new Eppendorftube, washed once with 70 percent ethanol, and finally washed with 100percent ethanol. The DNA was dried in vacuo for 1 minute and dissolvedin 0.75 mL deionized water. A 20 μL aliquot was diluted to 1.0 mL andthe OD 260 nm value was measured and recorded. The concentration of DNAin the original solution was calculated to be 0.30 mg/mL.

Oligonucleotides (oligos) were synthesized using phosphoramiditechemistry on a 380A DNA synthesizer (Applied Biosystems, Foster, Calif.)starting on 0.2 μM solid support columns. Protecting groups on the finalproducts were removed by heating in concentrated ammonia solution at 55°C. for 12 hours. Crude mixtures of oligonucleotides (approximately 12 OD260 nm units) were applied to 16 percent polyacrylamide-urea gels andelectrophoresed. DNA in the gels was visualized by short wave UV light.Bands were cut out and the DNA eluted by heating the gel pieces to 65°C. for 2 hours. Final purification was achieved by application of theeluted DNA solution onto C-18 Sep-Pac™ columns (Millipore) and elutionof the bound oligonucleotide with a 60 percent methanol solution. Thepure DNA was dissolved in deionized, distilled water (ddH₂ O) andquantitated by measuring OD 260 nm.

A GeneAmp™ DNA amplification kit (Cetus Corp., Emeryville, Calif.) wasused to clone the Hum4 V_(L) germline gene by the polymerase chainreaction (PCR), which was set up according to the manufacturer'sdirections. A thermal cycler was used for the denaturation (94° C.),annealing (45° C.) and elongation (72° C.) steps. Each of the threesteps in a cycle was carried out for 4 minutes; there was a total of 30cycles.

Upstream of the regulatory sequences in the Hum4 V_(L) germline gene,there is a unique Cla I restriction enzyme site. Therefore, the 5' endoligonucleotide for the PCR, called HUMVL(+) (FIG. 8A), was designed toinclude this Cla I site.

FIG. 9 shows the human J4 (HJ4) amino acid and DNA sequences. The firsttwo amino acids (Leu-Thr) complete the CDR3 region, the remainder makeup the FR4 region. Glu is underlined in HJ4 because in CC49 J5 a somaticmutation had occurred in this codon converting GAG (for Glu) to GTG (forVal). The (.arrow-down dbl.) indicates the slice site and the beginningof the intron between the J and C_(K) exons. DNA sequences underlined inHJ4 represent parts of the sequence used for the 3' end PCR oligo.

FIG. 10 is the DNA and amino acid sequence of Hum4 V_(L) inhuman/chimeric CC49H and CC83H. Specifically, the figure shows theentire DNA sequence of the Hum4 V_(L) gene Cla I-Hind III segment inpRL1001, clone #2. A single base difference occurred at position 3461and is marked by an asterisk (*). The corresponding amino acid sequencesin the coding exons are shown. The site of the Leu Pro mutation in clone#7 is boxed. An arrow (.Arrow-up bold.) indicates the site of the singlebase deletion in clone #11. The coding strand is underlined to designatethe sites used for hybridization of complementary oligonucleotideprimers. In order the primers occur from the 5' end as follows:HUMLIN1(-); HUMLIN2(-); HUMLCDR1(-) and Hind III C_(K) (-) (not shown).

The 3' end oligonucleotide, called HUMVL(-) (FIG. 8B), contained aunique Hind III site; sufficient mouse intron sequence past the splicingsite to permit an effective splice donor function; a human J4 sequencecontiguous with the 3' end of the V_(L) exon of Hum4 V_(L) to completethe CDR3 and FR4 sequences of the V_(L) domain (see FIGS. 9 and 10);nucleotides to encode a tyrosine residue at position 94 in CDR3; and 29nucleotides close to the 3' end of the V_(L) exon of Hum4 V_(L) (shownunderlined in the oligonucleotide HUMVL(-) in FIG. 8) to anneal with thehuman DNA target. In total, this 3' end oligonucleotide for the PCR was98 bases long with a non-annealing segment (a "wagging tail") of 69nucleotides. A schematic of the Hum4 V_(L) gene target and theoligonucleotides used for the PCR are shown in FIG. 11. A 5'-end oligo(HUMV_(L) (+)) and the 3'-end oligo (HUMV_(L) (-)) used to prime theelongation reactions for Taq polymerase and the target Hum4 V_(L) geneare shown.

A PCR reaction was set up with 1 μg of total human DNA in a reactionvolume of 100 μL. Primers HUMVL(-) and HUMVL(+) were each present at aninitial concentration of 100 pmol. Prior to the addition of Taqpolymerase (2.5 units/reaction) 100 μLs of mineral oil were used tooverlay the samples. Control samples were set up as outlined below. Thesamples were heated to 95° C. for 3 minutes. When the PCR was complete,20 μL samples were removed for analysis by agarose gel electrophoresis.

Based on the known size of the Hum4 V_(L) DNA fragment to be cloned, andthe size of the oligonucleotides used to target the gene, a product of1099 bp was expected. A band corresponding to this size was obtained inthe reaction (shown in lane 7, FIG. 12). Agarose gel electrophoresis ofHum4V_(L) PCR reactions. Lane 1: λHind III standard; lane 2:P no Taqpolymerase control; lane 3: no primers added; lane 4: no human DNAtemplate; lane 5: Gene Ampkit positive control; lane 6: 3 μg human DNAwith primers and Taq polymerase; lane 7: same as lane 6, but with lughuman DNA and lane 8: .O slashed.X174-HaeIII DNA standard. Ethidiumbromide was added to the gel and buffer. Bands were visualized by longwavelength UV light.

To prepare a plasmid suitable for cloning and subsequently expressingthe Hum4 V_(L) gene, the plasmid pSV2neo was obtained from ATCC andsubsequently modified. pSV2neo was modified as set forth below (seeFIGS. 13A-B).

The preparation of pSV2neo-101 was as follows. Ten micrograms ofpurified pSV2neo were digested with 40 units of Hind III at 37° C. for 1hour. The linearized plasmid DNA was precipitated with ethanol, washed,dried and dissolved in 10 μL of water. Two microliters each of 10 mMdATP, dCTP, dGTP and dTTP were added, as well as 2 μL of 10×ligasebuffer (Stratagene, La Jolla, Calif.). Five units (1 μL) of DNApolymerase I were added to make blunt the Hind III sticky ends. Thereaction mixture was incubated at room temperature for 30 minutes. Theenzyme was inactivated by heating the mixture to 65° C. for 15 minutes.The reaction mixture was then phenol extracted and ethanol precipitatedinto a pellet. The pellet was dissolved in 20 μL deionized, distilledwater. A 2 μL aliquot (ca. 1 μg) was then added to a standard 20 μLligation reaction, and incubated overnight at 4° C.

Competent E.coli DH1 cells (Invitrogen) were transformed with 1 μL and10 μL aliquots of a ligation mix (Invitrogen, San Diego, Calif.)according to the manufacturer's directions. Ampicillin resistantcolonies were obtained on LB plates containing 100 μg/mL ampicillin.Selected clones grown in 2.0 mL overnight cultures were prepared,samples of plasmid DNA were digested with Hind III and Bam HIseparately, and a correct representative clone selected.

The resulting plasmid pSV2neo-101 was verified by size mapping and thelack of digestion with Hind III.

A sample of DNA (10 μg) from pSV2neo-101 minilysate was prepared bydigesting with 50 units of Bam HI at 37° C. for 2 hours. The linearizedplasmid was purified from a 4 percent polyacrylamide gel byelectroelution. The DNA ends were made blunt by filling in the Bam HIsite using dNTPs and Klenow fragment, as described earlier for the HindIII site of pSV2 neo-101.

A polylinker segment containing multiple cloning sites was incorporatedat the Bam HI site of pSV2neo-101 to create pSV2neo-102, as shown inFIG. 14. The arrow (←) indicates the direction of the Eco RI site in thevector. Note that the polylinker could be inserted in both orientationssuch that the Barn HI site on the left side could also be regenerated.The nucleotides used to fill-in the Bam HI site are shown in italics.The top synthetic oligo was called (CH(+) while the complimentary strandwas CH(-). Equimolar amounts of two oligonucleotides, CH(+) and CH(-)(shown in FIG. 14) were annealed by heating for 3 minutes at 90° C. andcooling to 50° C. Annealed linker DNA and blunt ended pSV2neo-101 wereadded, in a 40:1 molar volume, to a standard 20 μL ligation reaction. E.coli DH1 (Invitrogen) was transformed with 0.5 μL and 5 μL aliquots ofthe ligation mixture (Invitrogen). Twelve ampicillin resistant colonieswere selected for analysis of plasmid DNA to determine whether thelinker had been incorporated.

A Hind III digest of mini-lysate plasmid DNA revealed linkerincorporation in six of the clones. The plasmid DNA from several cloneswas sequenced, to determine the number of linker units that wereblunt-end ligated to pSV2neo-101 as well as the relative orientation(s)with the linker. Clones for sequencing were selected on the basis ofpositive digestion with Hind III.

A Sequenase sequencing kit (United States Biochemical Corp, Cleveland,Ohio) was used to sequence the DNA. A primer, NE0102SEQ, was used forsequencing and is shown in FIG. 15. It is complementary to a sequencelocated upstream from the BamHI site in the vector. The Bam HI sitewhere the polylinker was inserted in pSV2neo-101 is boxed. Between 3 μgand 5 μg of plasmid DNA isolated from E.coli mini-lysates were used forsequencing. The DNA was denatured and precipitated prior to annealing,as according to the manufacturer's instructions. Electrophoresis wascarried out at 1500 volts; gels were dried prior to exposure to KodakX-ray film. Data was processed using a DNASIS™ computer program(Hitachi).

From the DNA sequence data of 4 clones analyzed (see photograph ofautoradiogram representing the sequence data of 2 of these clones- FIG.16, reading the sequence (going up) is in the 5' to 3' direction of the(+) strand), compared to the expected sequence in FIG. 14, two cloneshaving the desired orientation were obtained. In both cases of FIG. 16,a single 30-base linker unit was incorporated, but in oppositeorientations. Panel A-Sequence resulting in pSV2neo-120; PanelB-sequencing resulting in pSVneo-102. Reading the sequence (going up) isin the 5' to 3' direction of the (+) strand. In both cases a single30-base linker unit was incorporated, but in opposite orientations. Thepanel A-sequence resulted in pSV2neo-120; and the panel B sequenceresulted in pSV2neo-102. A representative clone was selected anddesignated pSV2neo-102.

A human CK gene was inserted into pSV2neo-102 to form pRL1000. The humanC_(K) DNA was contained in a 5.0 kb Hind III-Bam HI fragment (see Hieteret al. (1980), Cell, 22:197-207).

A 3 μg sample of DNA from a mini-lysate of pSV2neo-102 was digested withBam HI and Hind III. The vector DNA was separated from the small BamHI-Hind III linker fragment, generated in the reaction, byelectrophoresis on a 3.75 percent DNA polyacrylamide gel. The desiredDNA fragment was recovered by electroelution. A pBR322 clone containingthe 5.0 kb Hind III-Bam HI fragment of the human C_(K) gene (see Hieteret al., supra) was designated phumCK. The 5.0 kb Hind III-Bam HIfragment was ligated with pSV2neo-102 and introduced into E.coli DH1(Invitrogen). Ampicillin resistant colonies were screened and a clonecontaining the human C_(K) gene was designated pRL1000.

Finally, pRL1000 clones were screened by testing mini-lysate plasmid DNAfrom E. coli with HindIII and Bam HI. A clone producing a plasmid whichgave 2 bands, one at 5.8 kb (representing the vector) and the other at5.0 kb (representing the human C_(K) insert) was selected. Furthercharacterization of pRL1000 was achieved by sequencing downstream fromthe Hind III site in the intron region of the human C_(K) insert. Theoligonucleotide used to prime the sequencing reaction was NE0102SEQ (seeFIG. 15). Oligonucleotide synthesized (21-mer, called NE0102SEQ) tosequence putative pSV2neo-102 clones is the underlined sequence shownabove. The Bam HI site where the polylinker was inserted in pSVneo-101is boxed.

Two hundred and seventeen bases were determined (see FIG. 17). FIG. 17shows a DNA sequence form pRL1000, reading the (+) strand from theprimer NE0102SEQ (FIG. 15). Sequence data past the HindIII site is fromthe human C_(K) Hind III - Bam HI insert. The sequences complementary tothe underlined DNA sequences, called C_(K) (-), was synthesized as aprimer for sequencing in the upstream 3' direction. A newoligonucleotide corresponding to the (-) strand near the Hind III site(shown in FIG. 17) was synthesized so that clones, containing the Hum4V_(L) gene that were cloned into the Cla I and Hind III sites in pRL1000(see FIG. 13), could be sequenced.

A Cla I-Hind III DNA fragment containing Hum4 V_(L) obtained by PCR wascloned into the plasmid vector pRL1000. DNA of pRL1000 and the Hum4V_(L) were treated with Cla I and Hind III and the fragments were gelpurified by electrophoresis, as described earlier.

The pRL1000 DNA fragment and fragment containing Hum4 V_(L) gene wereligated, and the ligation mixture used to transform E.coli DH1(Invitrogen), following the manufacturer's protocol. Ampicillinresistant clones were screened for the presence of the Hum4 V_(L) geneby restriction enzyme analysis and a representative clone designatedpRL1001 (shown in FIG. 18). FIG. 18 shows a partial restriction map ofthe plasmid pRL1001. This is the expression vector to introduce thehuman anti-tumor L chain gene in Sp2/0 cells.

Four plasmids having the correct Cla I-Hind III restriction pattern wereanalyzed further by DNA sequencing of the insert region (see FIG. 19).FIG. 19 shows a DNA sequence autoradiogram of pRL1001 clones. Readingthe gel is in the 5' to 3' direction on the (-) strand, from the HindIII C_(K) (-) primer. Clones 2 and 9 were equivalent to the expectedsequence, clone 7 had a single base substitution (marked by *) and clone11 had a single base deletion (marked by -). Hind III C_(K) (-) (shownby underlining on the plus strand to which it hybridizes in FIG. 17),HUMLIN1(-) (shown by underlining on the plus strand to which ithybridizes in FIG. 10), HUMLIN2(-) (shown by underlining on the plusstrand to which it hybridizes in FIG. 10) and HUMLCDR1(-) (shown byunderlining on the plus strand to which it hybridizes in FIG. 10) wereused as the sequencing primers. Two out of the four plasmids analyzedhad the expected sequence in the coding regions (FIG. 19, clones 2 and9). The gel is read in the 5' to 3' direction on the (-) strand, fromthe Hind III C_(K) (-) primer. Clones 2 and 9 were equivalent to theexpected sequence, clone 7 had a single base substitution (marked by *)and clone 11 had a single base deletion (marked by →).

Clone 2 was chosen and used for generating sufficient plasmid DNA forcell transformations and other analysis. This plasmid was used forsequencing through the Hum4 V_(L), and the upstream region to the Cla Isite. Only one change at nucleotide position 83 from a C to a G (FIG.10) was observed, compared to a published sequence (Klobeck et al.(1985), supra). The DNA sequence data also indicates that theoligonucleotides used for PCR had been correctly incorporated into thetarget sequence.

A Biorad Gene Pulserr™ apparatus was used to transfect Sp2/0 cells withlinearized plasmid DNAs containing the light or heavy chain constructs.The Hum4 V_(L) was introduced into Sp2/0 cells along with correspondingheavy chains by the co-transfection scheme indicated in Table 1.

                  TABLE 1                                                         ______________________________________                                                 DNA Added                                                                                    H Chain H Chain                                         Cell Line L Chain p 49 p 83                                                   Designation pRL1001 gl-2.3 gl-2.3                                           ______________________________________                                        MP1-44H    20 μg     15 μg                                                                               0 μg                                        MP1-84H 20 μg  0 μg 15 ug                                             ______________________________________                                    

A total of 8.0×10⁶ Sp2/0 cells were washed in sterile PBS buffer (0.8 mLat 1×10⁷ viable cells/mL) and held on ice for 10 minutes. DNA ofpRL1001, linearized at the Cla I site, and DNA of either p49 g1-2.3 orp83 g1-2.3, linearized at their respective Nde I sites, were added, insterile PBS, to the cells (see protocol--Table 2) and held at 0° C. fora further 10 minutes. A single 200 volt, 960 μF electrical pulse lastingbetween 20 and 30 milliseconds was used for the electroporation. Afterholding the perturbed cells on ice for 5 minutes, 25 mL of RPMI mediumwith 10 percent fet al calf serum were introduced, and 1.0 mL samplesaliquoted in a 24 well tissue culture plate. The cells were incubated at37° C. in a 5 percent CO₂ atmosphere. After 48 hours, the media wasexchanged with fresh selection media, now containing both 1 mg/mLGeneticin (G418) (Difco) and 0.3 μg/ml mycophenolic acid/gpt medium.Resistant cells were cultured for between 7 and 10 days.

Supernatants from wells having drug resistant colonies were tested onELISA plates for activity against TAG-72. A roughly 10 percent pureTAG-72 solution prepared from LSI74T tumor xenograft cells was diluted1:40 and used to coat flexible polyvinyl chloride microtitration plates(Dynatech Laboratories, Inc.). Wells were air-dried overnight, andblocked the next day with 1 percent BSA. Supernatant samples to betested for anti-TAG-72 antibody were added to the washed wells andincubated for between 1 and 2 hours at 37° C. Alkaline phosphataselabeled goat anti-human IgG (diluted 1:250) (Southern BiotechAssociates, Birmingham, Ala.) was used as the probe antibody. Incubationwas for 1 hour. The substrate used was p-nitrophenylphosphate. Colordevelopment was terminated by the addition of 1.0 N NaOH. The plateswere read spectrophotometrically at 405 nm and 450 nm, and the valuesobtained were 405 nm-450 nm.

Those samples producing high values in the assay were suboloned from theoriginal 24 well plate onto 96 well plates. Plating was done at a celldensity of half a cell per well (nominally 50 cells) to get puremonoclonal cell lines. Antibody producing cell lines were frozen down inmedia containing 10 percent DMSO.

Two cell lines were procured having the designations: MP1-44H andMP1-84H. MP1-44H has the chimeric CC49 γ1 heavy chain with the Hum4V_(L) light chain; and MP1-84H has the chimeric CC83 γ1 heavy chain withthe Hum4 V_(L) light chain.

A 1.0 L spinner culture of the cell line MP1-44H was grown at 37° C. for5 days for antibody production. The culture supernatant was obtainedfree of cells by centrifugation and filtration through a 0.22 micronfilter apparatus. The clarified supernatant was passed over a Protein Acartridge (Nygene, N.Y.). Immunoglobulin was eluted using 0.1 M sodiumcitrate buffer, pH 3.0. The pH of the eluting fractions containing theantibody was raised to neutrality by the addition of Tris base, pH 9.0.The antibody-containing fractions were concentrated and passed over aPharmacia Superose 12 HR 10/30 gel filtration column. A protein wasjudged to be homogeneous by SDS polyacrylamide gel electrophoresis.Isoelectric focusing further demonstrated the purity of MP1-44H.

The biological performance of the human composite antibody, MP1-44H, wasevaluated by comparing immunohistochemistry results with two otheranti-TAG-72 antibodies CC49 (ATCC No. HB 9459) and Ch44 (ATCC No. HB9884). Sections of human colorectal tumor embedded in paraffin weretested with the three antibodies by methods familiar to those skilled inthis art. All three antibodies gave roughly equivalent bindingrecognition of the tumor antigen present on the tumor tissue sample.

A further test of the affinity and biological integrity of the humancomposite antibody MP1-44H was a competition assay, based oncross-competing radioiodine-labeled versions of the antibody with CC49and Ch44 in all combinations. From the data shown in FIGS. 23A-B, it isapparent that the affinity of all 3 antibodies is equivalent and canbind effectively to tumor antigen.

MP1-44H (ATCC HB 10426) and MP1-84H (ATCC HB 10427) were deposited atthe American Type Culture Collection (ATCC). The contract with ATCCprovides for permanent availability of the cell lines to the public onthe issuance of the U.S. patent describing and identifying the depositor the publications or upon the laying open to the public of any U.S. orforeign patent application, whichever comes first, and for availabilityof the cell line to one determined by the U.S. Commissioner of Patentsand Trademarks to be entitled thereto according to 35 CFR §122 and theCommissioner's rules pursuant thereto (including 37 CFR §1.14 withparticular reference to 886 OG 638). The assignee of the presentapplication has agreed that if the cell lines on deposit should die orbe lost or destroyed when cultivated under suitable conditions for aperiod of thirty (30) years or five (5) years after the last request, itwill be promptly replaced on notification with viable replacement celllines.

Example 2

Single-chain antibodies consist of a V_(L), V_(H) and a peptide linkerjoining the V_(L) and V_(H) domains to produce SCFVs. A single chainantibody, SCFV1, was constructed to have the Hum4 V_(L) as V Domain 1and CC49 V_(H) as V Domain 2 (see FIG. 21).

The polypeptide linker which joins the two V domains was encoded by DNAintroduced at the 3' end of the Hum4 V_(L) DNA during a PCR. Theoligonucleotides SCFV1a and SCFV2 were designed to obtain the DNAsegment incorporating part of the yeast invertase leader sequence, theHum4 V_(L) and the SCFV linker.

The polypeptide linker for SCFV1 was encoded in oligonucleotide SCFV1b(see below). The underlined portions of the oligonucleotides SCFV1a andSCFV1b are complementary to sequences in the Hum4 V_(L) and linkerrespectively. The sequences of SCFV1a and SCFV1b are as follows, withthe hybridizing sequences underlined:

SCFV1a with the Hind III in bold:

5'-CTGCAAGCTTCCTTTTCCTTTTGGCTGGTTTTGCAGCCAAAATATCTGCAGACATCGTGATGACCCAGTC-3'

SCFV1b with the Aat II site in bold:

5'-CGTAAGACGTCTAAGGAACGAAATTGGGCCAATTGTTCTGAGGAGACCGAACCTGACTCCTTCACCTTGGTCCCTCCGCCG-3'

The target DNA in the PCR was pRL1001 (shown in FIG. 18). The PCR wasperformed pursuant to the teachings of Mullis et al., supra. A DNAfragment containing the Hum4 V_(L) -linker DNA component for theconstruction of SCFV1 was obtained and purified by polyacrylamide gelelectrophoresis according to the teachings of Sambrook et al., supra.

p49 g1-2.3, containing CC49 V_(H), was the target DNA in the PCR. PCRwas performed according to the methods of Mullis et al., supra. Theoligonucleotides used for the PCR of CC49 V_(H) are as follows, with thehybridizing sequences underlined:

SCFV1c, with the Aat II site in bold:

5'-CCTTAGACGTCCAGTTGCAGCAGTCTGACGC-3'

SCFV1d, with the Hind III site in bold:

5'-GATCAAGCTTCACTAGGAGACGGTGACTGAGGTTCC-3'

The purified Hum4 V_(L) -linker and V_(H) DNA fragments were treatedwith Aat II (New England Biolabs, Beverly, Mass.) according to themanufacturer's protocol, and purified from a 5 percent polyacrylamidegel after electrophoresis. An equimolar mixture of the Aat II fragmentswas ligated overnight. The T4 DNA ligase was heat inactivated by heatingthe ligation reaction mixture at 65° C. for 10 minutes. Sodium chloridewas added to the mixture to give a final concentration of 50 mM and themixture was further treated with Hind III. A Hind III DNA fragment wasisolated and purified from a 4.5 percent polyacrylamide gel and clonedinto a yeast expression vector (see Carter et al. (1987), In: DNACloning, A Practical Approach, Glover (ed.) Vol III: 141-161). Thesequence of the fragment, containing the contiguous SCFV1 construct, isset forth in FIG. 22.

The anti-TAG-72 SCFV1 described herein utilized the yeast invertaseleader sequence (shown as positions -19 to -1 of FIG. 22), the Hum4V_(L) (shown as positions 1 to 113 of FIG. 22), an 18 amino acid linker(shown as positions 114 to 132 of FIG. 22) and CC49 V_(H) (shown aspositions 133 to 248 of FIG. 22).

The complete DNA and amino acid sequence of SCFV1 is given in FIG. 22.The oligonucleotides used to sequence the SCFV1 are set forth below.

TPI:

5'-CAATTTTTTGTTTGTATTCTTTTC-3'.

HUVKF3:

5'-CCTGACCGATTCAGTGGCAG-3'.

DC113:

5'-TCCAATCCATTCCAGGCCCTGTTCAGG-3'.

SUC2T:

5'-CTTGAACAAAGTGATAAGTC-3'.

Example 3

A plasmid, pCGS517 (FIG. 23), containing a prorennin gene was digestedwith Hind III and a 6.5 kb fragment was isolated. The plasmid pCGS517has a triosephosphate isomerase promoter, invertase [SUC2] signalsequence, the prorennin gene and a [SUC2] terminator. The HindIII-digested SCFV1 insert obtained above (see FIG. 23) was ligatedovernight with the Hind III fragment of pCGS517 using T4 DNA ligase(Stratagene, La Jolla, Calif.).

The correct orientation existed when the Hind III site of the insertcontaining part of the invertase signal sequence ligated to the vectorDNA to form a gene with a contiguous signal sequence. E.coli DHI(Invitrogen) cells were transformed and colonies screened using afilter-microwave technique (see Buluwela, et al. (1989), Nucleic AcidsResearch, 17:452). From a transformation plate having several hundredcolonies, 3 positive clones were obtained. Digesting the candidateplasmids with Sal I and Kpn I, each a single cutter, differentiatedbetween orientations by the size of the DNA fragments produced. A singleclone, pDYSCFV1 (FIG. 23), had the correct orientation and was used forfurther experimentation and cloning. The probe used was derived frompRL1001, which had been digested with Kpn I and Cla I (see FIG. 18). Theprobe DNA was labeled with ³² P α-dCTP using a random oligonucleotideprimer labeling kit (Pharmacia LKB Biotechnology, Piscataway, N.J.).

The next step was to introduce the Bgl II-Sal 1 fragment from pDYSCFV1into the same restriction sites of another vector (ca. 9 kb), which wasderived from PCG5515 (FIG. 23), to give an autonomously replicatingplasmid in S. cerevisiae.

DNA from the vector and insert were digested in separate reactions withBgl II and Sal I using 10×buffer number 3 (50 MM Tris-HCl (pH 8.0), 100mM NaCl, BRL). The DNA fragment from pDYSCFV1 was run in andelectroeluted from a 5 percent polyacrylamide gel and the insert DNA wasrun and electroeluted from a 3.75 percent polyacrylamide gel. A standardligation using T4 DNA ligase (Stratagene, La Jolla, Calif.) and atransformation using E.coli DH1 (Invitrogen) was carried out. Out of 6clones selected for screening with Bgl II and Sal I, all 6 werecorrectly oriented, and one was designated pCGS515/SCFV1 (FIGS. 23A-B).

DNA sequencing of pCGS515/SCFV1 DNA was done using a Sequenase™ kit(U.S. Biochemical, Cleveland, Ohio) using pCGS515/SCFV1 DNA. The resultshave been presented in FIG. 22 and confirm the sequence expected, basedon the linker, the Hum4 V_(L) and the CC49 V_(H).

Transformation of yeast cells using the autonomously replicating plasmidpCGS515/SCFV1 was carried out using the lithium acetate proceduresdescribed in Ito et al. (1983), J. Bacteriol., 153:163-168; and Treco(1987), In: Current Protocols in Molecular Biology, Ausebel et al.(eds), 2:13.71-13.7.6. The recipient strain of S.cerevisae was CGY1284having the genotype--MAT α (mating strain α), ura 3-52 (uracilauxotrophy), SSC1-1 (supersecreting 1), and PEP4⁺ (peptidase 4positive).

Transformed clones of CGY1284 carrying SCFV plasmids were selected bytheir ability to grow on minimal media in the absence of uracil.Transformed colonies appeared within 3 to 5 days. The colonies weretransferred, grown and plated in YEPD medium. Shake flasks were used toprovide culture supernatant with expressed product.

An ELISA procedure was used to detect biological activity of the SCFV1.The assay was set up such that the SCFV would compete with biotinylatedCC49 (biotin-CC49) for binding to the TAG-72 antigen on the ELISA plate.

SCFV1 protein was partially purified from a crude yeast culturesupernatant, using a Superose 12 gel filtration column (Pharmacia LKBBiotechnology), and found to compete with biotinylated CC49 in thecompetition ELISA. These results demonstrate that the SCFV1 had TAG-72binding activity.

The SCFV1 protein was detected by a standard Western protocol (seeTowbin et al. (1979), Proc. Natl. Acad. Sci., USA, 76:4350-4354). Thedetecting agent was biotinylated FAID14 (ATCC No. CRL 10256), ananti-idiotypic monoclonal antibody prepared from mice that had beenimmunized with CC49. A band was visualized that had an apparentmolecular weight of approximately 26,000 daltons, the expected size ofSCFV1. This result demonstrated that the SCFV1 had been secreted andproperly processed.

Example 4

The following example demonstrates the cloning of human V_(H) genes intoa SCFV plasmid construct containing sequence coding for the Hum4 V_(L)and a 25 amino acid linker called UNIHOPE.

A vector was prepared from plasmid pRW 83 containing a chloramphenicolresistance (Cam^(r)) gene for clone selection, and a penP gene with apenP promoter and terminator (see Mezes, et al. (1983), J. Biol. Chem.,258:11211-11218) and the pel B signal sequence (see Lei, et al. (1987),supra). The vector was designated Fragment A (see FIG. 24). The penPgene was removed with a Hind III/Sal I digest.

The penP promoter and pel B signal sequence were obtained by a PCR usingpRW 83 as a template and oligonucleotides penP1 and penP2 as primers.The fragment was designated Fragment B (see FIG. 24). A Nco I enzymerestriction site was introduced at the ₃ ' end of the signal sequenceregion by the penP2 oligonucleotide.

penP1:

5'-CGATAAGCTTGAATTCCATCACTTCC-3'

penP2:

5'-GGCCATGGCTGGTTGGGCAGCGAGTAATAACAATCCAGCG GCTGCCGTAGGCAATAGGTATTTCATCAAAATCGTCTCCCTCCGTTTGAA-3'

A SCFV comprised of a Hum4 V_(L), a CC49 V_(H), and an 18 amino acidlinker (Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg SerLeu Asp) was obtained from pCGS515/SCFV1 by PCR using oligonucleotidespenP3 and penP6. This fragment was designated Fragment D (see FIG. 24).A Bcl I site was introduced at the 3' end of the V_(H) region by thepenP6 oligonucleotide.

penP3:

5'-GCTGCCCAACCAGCCATGGCCGACATCGTGATGACCCAGTCTCC-3'

penP6(-):

5'-CTCTTGATCACCAAGTGACTTTATGTAAGATGATGTTTTG ACGGATTCATCGCAATGTTTTTATTTGCCGGAGACGGTGACTGAGGTTCC-3'.

Fragments B and D were joined by PCR using oligonucleotides penP1 andpenP6, following the procedures of Horton et al., supra. The newfragment was designated E (See FIG. 24).

Fragment C containing the penP termination codon was isolated bydigesting pRW 83 with Bcl I and Sal I, and designated Fragment C. pRW 83was isolated from E. coli strain GM161, which is DNA methylase minus ordam⁻.

Plasmid pSCFV 31 (see FIG. 24) was created with a three part ligationFragments A, C, and E.

The Nco I restriction enzyme site within the Cam^(r) gene and the HindIII site located at the 5' end of the penp promoter in pSCFV 31 weredestroyed through a PCR DNA amplification using oligonucleotides Nco1.1and Nco1.3(-) to generate an Eco RI-Nco I fragment and oligonucleotidesNco1.2 and Nco1.4c(-) to generate a Nco I to Eco RI fragment. These twofragments were joined by PCR-SOE using oligonucleotides Nco1.1 andNco1.4c(-). The oligonucleotides are set forth below:

Nco1.1:

5'-TCCGGAATTCCGTATGGCAATGA-3'

Nco1.3(-):

5'-CTTGCGTATAATATTTGCCCATCGTGAAAACGGGGGC-3'

Nco1.2:

5'-ATGGGCAAATATTATACGCAAG-3'

Nco1.4c(-):

5'-CACTGAATTCATCGATGATAAGCTGTCAAACATGAG-3'

pSCFV 31 was digested with Eco RI and the larger fragment was isolatedby polyacrylamide gel electrophoresis. To prevent self ligation, the DNAwas dephosphorylated using calf intestinal alkaline phosphataseaccording to the teachings of Sambrook et al., supra.

A two part ligation of the larger pSCFV 31 digested fragment and thePCR-SOE fragment, described above, resulted in the creation of pSCFV 31b(see FIG. 25).

pSCFV 31b was digested with Nco I and Sal I and a fragment containingthe Cam^(r) gene was isolated.

The Hum4 V_(L) was obtained by PCR DNA amplification using pCGS515/SCFV1as a template and oligonucleotides 104BH1 and 104BH2(-) as primers.

104BH1:

5'-CAGCCATGGCCGACATCGTGATGACCCAGTCTCCA-3'

104BH2(-):

5'-AAGCTTGCCCCATGCTGCTTTAACGTTAGTTTTATCTGCTGGAGACAGAGTGCCTTCTGCCTCCACCTTGGTCCCTCCGCCGAAAG-3'

The CC49 V_(H) was obtained by PCR using p49 g1-2.3 (FIG. 6) as atemplate and oligonucleotides 104B3 and 104B4(-) as primers. A Nhe Ienzyme restriction site was introduced just past the termination codonin the 3' end (before the Bcl I site) by oligonucleotide 104B4(-).

104B3:

5'-GTTAAAGCAGCATGGGGCAAGCTTATGACTCAGTTGCAGCAGTCTGACGC-3'

104B4(-):

5'-CTCTTGATCACCAAGTGACTTTATGTAAGATGATGTTTTGACGGATTCATCGCTAGCTTTTTATTTGCCATAATAAGGGGAGACGGTGACTGAGGTTCC-3'

In the PCR which joined these two fragments using oligonucleotides104BH1 and 104B4(-) as primers, a coding region for a 22 amino acidlinker was formed.

A fragment C (same as above) containing the penP termination codon wasisolated from pRW 83 digested with Bcl I and Sal I.

Plasmid pSCFV 33H (see FIGS. 25A-B) was created with a three partligation of the vector, fragment C, and the SCFV fragment describedabove.

pSCFV 33H was digested with NcoI and NheI, and the DNA fragmentcontaining the Cam^(r) gene was isolated as a vector.

Hum4 V_(L) was obtained by PCR DNA amplification using pRL1001 (see FIG.18) as a template and oligonucleotides UNIH1 and UNIH2(-) as primers.Oligonucleotides for the PCR were:

UNIH1:

5'-CAGCCATGGCCGACATTGTGATGTCACAGTCTCC-3'

The Nco I site is in bold and the hybridizing sequence is underlined.

UNIH2(-):

5'-GAGGTCCGTAAGATCTGCCTCGCTACCTAGCAAAAGGTCCTCAAGCTTGATCACCACCTTGGTCCCTCCGC-3'

The Hind III site is in bold.

The CC49 V_(H) was obtained by a PCR using p49g1-2.3 (see FIG. 6) as atemplate and oligonucleotides UNI3 and UNI4(-) as primers.

UNI3:

5'-AGCGAGGCAGATCTTACGGACCTCGAGGTTCAGTTGCAGCAGTCTGAC-3'

The Xho I site is in bold and the hybridizing sequence is underlined.

UNI4(-):

5'-CATCGCTAGCTTTTTATGAGGAGACGGTGACTGAGGTTCC-3'.

The Nhe I site is in bold and the hybridizing sequence is underlined.

Oligonucleotides UNIH1 and UNI4(-) were used in the PCR-SOEamplification which joined the Hum4 V_(L) and CC49 V_(H) fragments andformed a coding region for a negatively charged fifteen amino acidlinker. The DNA was digested with Nhe I and Nco I and ligated with thevector fragment from the Nco I-Nhe I digest of pSCFV 33H. The resultantplasmid was designated pSCFV UNIH (shown in FIG. 25).

With the construction of pSCFV UNIH, a universal vector for any SCFV wascreated with all the desired restriction enzyme sites in place.

pSCFV UNIH was digested with Hind III/Xho I, and the large DNA fragmentcontaining the Cam^(r) gene, Hum4 V_(L) and CC49 V_(H) was isolated.

A fragment coding for a 25 amino acid linker, was made by annealing thetwo oligonucleotides shown below. The linker UNIHOPE is based on 205CSCA™ linker (see Whitlow, (1990) Antibody Engineering: New Technologyand Application Implications, IBC USA Conferences Inc, MA), but thefirst amino acid was changed from serine to leucine and the twenty-fifthamino acid were was changed from glycine to leucine, to accommodate theHind III and Xho I restriction sites. The nucleotide sequence of thesingle chain portion of pSCFV Unihope H is shown in FIG. 26. Structuralsequences are indicated by the amino acid sequence written above the DNAsequence. The symbols → and ← indicate the beginning and end of a givensegment. The amino acid sequence of the linker is boxed.

The nucleotide sequence encoding the linker UNIHOPE is set forth below:

UNIHOPE (FIG. 26):

5'-TATAAAGCTTAGTGCGGACGATGCGAAAAAGGATGCTGCGAAGAAGGATGACGCTAAGAAAGACGATGCTAAAAAGGACCTCGAGTCTA-3'

UNIHOPE(-) (FIG. 26):

5'-TAGACTCGAGGTCCTTTTTAGCATCGTCTTTCTTAGCGTCATCCTTCTTCGCAGCATCCTTTTTCGCATCGTCCGCACTAAGCTTTATA-3'.

The resulting strand was digested with Hind IIIlXho I and ligated intothe vector. thus generating the plasmid pSCFV UHH (shown in FIG. 27).Plasmid pSCFV UHH expresses a biologically active, TAG-72 binding SCFVconsisting of the Hum4 V_(L) and CC49 V_(H). The expression plasmidutilizes the β-lactamase penP promoter, pectate lyase pelB signalsequence and the penP terminator region. Different immunoglobulin lightchain variable regions can be inserted in the Nco I-Hind III restrictionsites, different SCFV linkers can be inserted in the Hind III-Xho Isites and different immunoglobulin heavy chain variable regions can beinserted in the Xho I-Nhe I sites.

E.coli AG1 (Stratagene) was transformed with the ligation mix, and afterscreening, a single chloramphenicol resistant clone, having DNA with thecorrect restriction map, was used for further work.

The DNA sequence and deduced amino acid sequence of the SCFV gene in theresulting plasmid are shown in FIG. 26.

E.coli AG1 containing pSCFV UHH were grown in 2 ml of LB broth with 20μg/mL chloramphenicol (CAM 20). The culture was sonicated and assayedusing a competition ELISA. The cells were found to produce anti-TAG-72binding material. The competition assay was set up as follows: a 96 wellplate was derivatized with a TAG-72 preparation from LS174T cells. Theplate was blocked with 1% BSA in PBS for 1 hour at 31° C. and thenwashed 3 times. Twenty-five microliters of biotin CC49 (1/20,000dilution of a 1 mg/mL solution) were added to the wells along with 25 μLof sample to be tested (competition step) and the plate was incubatedfor 30 minutes at 31° C. The relative amounts of TAG-72 bound to theplate, biotinylated CC49, streptavidin-alkaline phosphatase, and colordevelopment times were determined empirically in order not to haveexcess of either antigen or biotinylated CC49, yet have enough signal todetect competition by SCFV. Positive controls were CC49 at 5 μg/mL andCC49 Fab at 10 μL/mL. Negative controls were 1% BSA in PBS and/orconcentrated LB. At the end of the competition step, unbound proteinswere washed away.

Fifty microliters of a 1:1000 dilution of streptavidin conjugated withalkaline phosphatase (Southern Biotechnology Associates, Inc.,Birmingham, Ala.) were added and the plate was incubated for 30 minutesat 31° C. The plate was washed 3 more times. Fifty microliters of apara-nitrophenylphosphate solution (Kirkegaard & Perry Laboratories,Inc., Gaithersburg, Md.) were added and the color reaction was allowedto develop for a minimum of 20 minutes. The relative amount of SCFVbinding was measured by optical density scanning at 405-450 nm using amicroplate reader (Molecular Devices Corporation, Menlo Park, Calif.).Binding of the SCFV resulted in decreased binding of the biotinylatedCC49 with a concomitant decrease in color development. The average valuefor triplicate test samples is shown in the table below:

    ______________________________________                                        Sample (50 μL) OD 405 nm - OD 450 nm Value                                   (mixed 1:1 with CC49 Biotin) at 50 minutes                                  ______________________________________                                        Sonicate E. coli AG1/ pSCFVUHH                                                                  0.072                                                         clone 10                                                                      Sonicate E. coli AG1/ pSCFVUHH 0.085                                          clone 11                                                                      CC49 at 5 mg/mL 0.076                                                         CC49 Fab at 10 mg/mL 0.078                                                    LB (negative control) 0.359                                                 ______________________________________                                    

The data indicates that there was anti-TAG-72 activity present in theE.coli AGI/pSCFVUHH clone sonicate.

Example 5

The plasmid pSCFVUHH may be used to host other V_(H) genes on Xho I-NheI fragments and test in a SCFV format, following the procedures setforth below. A schematic for this process is shown in FIG. 31.

Isolating total RNA from peripheral blood lymphocytes

Blood from a normal, healthy donor is drawn into three 5 mL purple-capVacutainer tubes. Seven mL of blood are added to two 15 mL polypropylenetubes. An equal volume of lymphoprep (cat#AN5501, Accurate) is added andthe solution is mixed by inversion. Both tubes are centrifuged at 1000rpm and 18° C. for 20 minutes. The resulting white area near the top ofthe liquid (area not containing red blood cells) is removed from eachsample and placed into two sterile polypropylene centrifuge tube. Ten mLof sterile PBS are added and the tube mixed by inversion. The samplesare centrifuged at 1500 rpm and 18° C. for 20 minutes Total RNA isisolated from resulting pellet according to the RNAzol B Method(Chomczynski and Sacchi (1987), Analytical Biochemistry, 162:156-159).Briefly, the cell pellets are lysed in 0.4 mL RNAzol solution(cat#:CS-105, Cinna/Biotecx). RNA is solubilized by passing the cellpellet through a 1 mL pipette tip. Sixty μL of chloroform are added andthe solution is shaken for 15 seconds. RNA solutions are then placed onice for 5 minutes. Phases are separated by centrifugation at 12000×g and4° C. for 15 minutes. The upper (aqueous) phases are transferred tofresh RNase-free microcentrifuge tubes. One volume of isopropanol isadded and the samples placed at -20° C. for 1 hour. The samples are thenplaced on dry ice for 5 minutes and finally centrifuged for 40 secondsat 14,000×g and 4° C. The resulting supernatant is removed from eachsample and the pellet is dissolved in 144 μL of sterile RNase-freewater. Final molarity is brought to 0.2 in NaCl. The DNA isreprecipitated by adding 2 volumes of 100% ethanol, leaving on dry icefor 10 minutes, and centrifugation at 14,000 rpm and 4° C. for 15minutes. The supernatants are then removed, the pellets washed with 75%ethanol and centrifuged for 8 minutes at 12000×g and 4° C. The ethanolis then removed and the pellets dried under vacuum. The resulting RNA isthen dissolved in 20 sterile water containing 1 μl RNasin (cat#:N2511,Promega).

cDNA synthesis:

cDNA synthesis is performed using a Gene Amp™ PCR kit (cat#: N808-0017Perkin Elmer Cetus), RNasin™ (cat#: N2511, Promega), and AMV reversetranscriptase (cat#: M9004, Promega). The following protocol is used foreach sample:

    ______________________________________                                        Components             Amount                                                 ______________________________________                                        MgCl.sub.2 solution    4 μl                                                  10 × 2 μl                                                            PCR buffer II                                                                 dATP 2 μl                                                                  dCTP 2 μl                                                                  dGTP 2 μl                                                                  dTTP 2 μl                                                                  3' primer 1 μl                                                             (random hexamers)                                                             RNA sample 2 μl                                                            RNasin 1 μl                                                                AMV RT 1.5 μl                                                            ______________________________________                                    

Samples are heated at 80° C. for 3 minutes then slowly cooled to 48° C.The samples are then centrifuged for 10 seconds. AMV reversetranscriptase is added to the samples which are then incubated for 30minutes at 37° C. After incubation, 0.5 μl of each dNTP and 0.75 reversetranscriptase (cat#:109118, Boehringer Mannheim) are added. The samplesare incubated for an additional 15 minutes at 37° C.

PCR Reaction:

Oligonucleotides are designed to amplify human V_(H) genes by polymerasechain reaction. The 5' oligonucleotides are set forth below:

HVH135:

5'-TATTCTCGAGGTGCA(AG)CTG(CG)TG(CG)AGTCTGG-3'

HVH2A:

5'-TATTCTCGAGGTCAA(CG)TT(AG)A(AG)GGAGTCTGG-3'

HVH46:

5'-TATTCTCGAGGTACAGCT(AG)CAG(CG)(AT)GTC(ACG)GG-3'

The 3' oligonucleotides are set forth below:

JH1245:

5'-TTATGCTAGCTGAGGAGAC(AG)GTGACCAGGG-3'

JH3:

5'-TTATGCTAGCTGAAGAGACGGTGACCATTG

JH6:

5'-TTATGCTAGCTGAGGAGACGGTGACCGTGG-3'

PCR reactions are performed with a GeneAmp™ PCR kit (cat#:N808-0017,Perkin Elmer Cetus). Components are listed below:

    ______________________________________                                        Components             Amount                                                 ______________________________________                                        ddH.sub.2 O            75 μl                                                 10 × buffer 10 μl                                                    dATP 2 μl                                                                  dCTP 2 μl                                                                  dGTP 2 μl                                                                  dTTP 2 μl                                                                  1* Target DNA 1 μl                                                         2* 5' primer 2.5 μl                                                        3' primer 2.0 μl                                                           3* AmpliTaq ™ 1.3 μl                                                    Polymerase                                                                  ______________________________________                                         *components added in order at 92° C. of first cycle               

PCR program:

step 1 94° C. for 30 seconds

step 2 60° C. for 1 minutes

step 3 72° C. for 45 seconds

Approximately 35 cycles are completed for each reaction. All PCRreactions are performed using a Perkin Elmer Cetus PCR System 9600thermal cycler.

Treatment of Human V_(H) inserts with Xho I and Nhe I

Human V_(H) genes are digested with Xho I (cat#: 131L, New EnglandBiolabs) and Nhe I (cat#: 146L, New England Biolabs). The followingprotocol is used for each sample:

    ______________________________________                                        SUBSTANCE             AMOUNT                                                  ______________________________________                                        DNA                   20 μl                                                  NEB Buffer #2 4.5 μl                                                       Nhe I 2 μl                                                                 Xho I 2 μl                                                                 ddH.sub.2 O 16.5 μl                                                      ______________________________________                                    

Samples are incubated at 37° C. for 1 hour. After this incubation, anadditional 1.5 μL Nhe I is added and samples are incubated an additionaltwo hours at 37° C.

Purification of DNA

After the restriction enzyme digest, DNA is run on a 5 percentpolyacrylamide gel (Sambrook et al. (1989), supra). Bands of 390-420 bpin size are excised from the gel. DNA is electroeluted and ethanolprecipitated according to standard procedures.

PCR products resulting from oligonucleotide combinations are pooledtogether: JH1245 with HVH135, HVH2A and HVH46; JH3 with HVH135, HVH2Aand HVH46; JH6 with HVH135, HVH2A and HVH46. The volume of the resultingpools are reduced under vacuum to 50 microliters. The pools are thenpurified from a 4 percent polyacrylamide gel (Sambrook et al. (1989),supra) to isolate DNA fragments. Bands resulting at 390-420 bp areexcised from the gel. The DNA from excised gel slices is electroelutedaccording to standard protocols set forth in Sambrook, supra.

Isolation of pSCFVUHH Xho I/Nhe I Vector Fragment

Approximately 5 μg in 15 μL of pSCFVUHH plasmid is isolated using theMagic Mini-prep™ system (Promega). To this is added 5.4 μL OF 10×Buffer#2 (New England Biolabs), 45 units of Xho I (New England Biolabs), 15units of Nhe I and 24 μL of ddH₂ O. The reaction is allowed to proceedfor 1 hour at 37° C. The sample is loaded on a 4% polyacrylamide gel,electrophoresed and purified by electroelution, as described earlier.The DNA pellet is dissolved in 20 μL of ddH₂ O.

One hundred nanograms of pSCFVUHH digested with Xho I/Nhe I is ligatedwith a 1:1 molar ratio of purified human V_(H) inserts digested with XhoI and Nhe I using T4 DNA ligase (Stratagene). Aliquots are used totransform competent E.coli AG1 cells (Stratagene) according to thesupplier's instructions.

GVWP hydrophilic membranes (cat#GVWP14250, Millipore) are placed on CAM20 LB agar plates (Sambrook et al., 1989). One membrane is added to eachplate. Four hundred microliters of the E.coli AG1 transformationsuspension from above are evenly spread over the surface of eachmembrane. The plates are incubated for 16 hours at 37° C.

Preparation of TAG-72-coated membranes

A 1% dilution of partially purified tumor associated glycoprotein-72(TAG-72) produced in LS174 T-cells is prepared in TBS (cat# 28376,Pierce). Ten milliliters of the TAG dilution are placed in a petri plate(cat# 8-757-14, Fisher) for future use. Immobilon-P PVDF transfermembranes (cat# SE151103, Millipore) are immersed in methanol. Themembranes are then rinsed three times in sterile double distilled water.After the final wash, the excess water is allowed to drain. Each of themembranes are placed in 10 milliliters of dilute TAG-72. The membranesare incubated at ambient temperature from 1 hour with gentle shaking.After incubation, the membranes are blocked with Western blockingsolution (25 mM Tris, 0.15 M NaCl, pH 7.6; 1% BSA) for about 1 hour atambient temperature.

Blocking solution is drained from the TAG membranes. With the sideexposed to TAG-72 facing up, the membranes are placed onto fresh CAM 20plates. Resulting air pockets are removed. The bacterial membranes arethen added, colony side up, to a TAG membrane. The agar plates areincubated for 24 to 96 hours at ambient temperatures.

The orientation of the TAG-72 and bacterial membranes are marked withpermanent ink. Both membranes are removed from the agar surface. TheTAG-72 membrane is placed in 20 ml of Western antibody buffer (TBS in0.05% Tween-20, cat# P-1379, Sigma Chemical Co.; 1% BSA, cat#3203,Biocell Laboratories) containing 0.2 ng of CC49-Biotin probe antibody.The bacterial membranes are replaced on the agar surface in theiroriginal orientation and set aside. CC49-Biotin is allowed to bind tothe TAG membranes for 1 hour at 31° C. with gentle shaking. Themembranes are then washed three times with TTBS (TBS, 0.05% Tween-20)for 5 minutes on an orbital shaker at 300 rpm. Streptavidin alkalinephosphatase (cat# 7100-04, Southern Biotechnology Associates) is addedto Western antibody buffer to produce a 0.1% solution. The TAG-72membranes are each immersed in 16 milliliters of the streptavidinsolution and allowed to incubate for 30 minutes at 31° C. with gentleshaking. After incubation, the membranes are washed as previouslydescribed. A final wash is then performed using Western alkalinephosphate buffer (8.4 g NaCO₃, 0.203 g MgCl₂ -H₂ O, pH 9.8), for 2minutes at 200 rpm at ambient temperature. To develop the membranes,Western blue stabilized substrate (cat# S384B, Promega) is added to eachmembrane surface. After 30 minutes at ambient temperatures, developmentof the membranes is stopped by rinsing the membranes three times withddH₂ O. The membranes are then photographed and clear zones arecorrelated with colonies on the hydrophilic membrane, set aside earlier.Colony(ies) are isolated for growth in culture and used to prepareplasmid DNA for sequencing characterization. Also, the protein productis isolated to evaluate specificity and affinity.

Identification of Hum4 V_(L), human V_(H) combinations using pATDFLAG

In a second assay system, Hum4 V_(L) --human V_(H) combinations arediscovered that bind to TAG-72 according to the schematic, supra, exceptfor the following a different plasmid vector, pATDFLAG, was used (seebelow): at the assay step, IBI MII antibody is used as a probe to detectany Hum4 V_(L) - V_(H) SCFV combinations that have bound to thehydrophobic membrane coated with TAG-72 and a sheep anti-mouse Igantibody conjugated to horseradish peroxidase (Amersham, ArlingtonHeights, Ill.) is used to detect any binding of the MII antibody toTAG-72.

The plasmid pATDFLAG was generated from pSCFVUHH (see FIGS. 29A-B) toincorporate a Flag-coding sequence 3' of any human V_(H) genes to beexpressed contiguously with Hum4 V_(L). The plasmid pATDFLAG, whendigested with Xho I and Nhe I and purified becomes the human V_(H)discovery plasmid containing Hum4 V_(L) in this SCFV format. The plasmidpATDFLAG was generated as follows. Plasmid pSCFVUHH treated with Xho Iand Nhe I (isolated and described above) was used in a ligation reactionwith the annealed FLAG and FLAGNC oligonucleotides.

FLAGC:

5'-TCGAGACAATGTCGCTAGCGACTACAAGGACGATGATGACAAATAAAAAC-3'

FLAGNC:

5'-CTAGGTTTTTATTTGTCATCATCGTCCTTGTAGTCGCTAGCGACATTGTC-3'

Equimolar amounts (1×10⁻¹⁰ moles of each of the oligonucleotides FLAGCand FLAGNC were mixed together using a ligation buffer (Stratagene). Thesample is heated to 94° C. and is allowed to cool to below 35° C. beforeuse in the ligation reaction below.

Ligation Reaction to Obtain pATDFLAG

    ______________________________________                                        COMPONENT              AMOUNT                                                 ______________________________________                                        pSCFVUHH Xho I/Nhe     1.5 μl                                                I vector                                                                      ANNEALED 0.85 μl                                                           FLAGC/FLAGNC                                                                  10 × Ligation 2 μl                                                   buffer                                                                        T4 DNA LIGASE 1 μl                                                         10 MM ATP 2 μl                                                             ddH.sub.2 O 12.65 μl                                                     ______________________________________                                    

This ligation reaction is carried out using the following components andamounts according the ligation protocol disclosed above. E.coli AG1cells (Stratagene) are transformed with 3 μl of the above ligationreaction and colonies selected using CAM 20 plates. Clones havingappropriate Nhe I, Xho I and Nhe IlXho I restriction patterns areselected for DNA sequencing.

The oligonucleotide used to verify the sequence of the FLAG linker inpATDFLAG (see FIG. 28) is called PENPTSEQ: 5'-CTTTATGTAAGATGATGTTTTG-3.This oligonucleotide is derived from the non-coding strand of the penPterminator region. DNA sequencing is performed using Sequenase™sequencing kit (U.S. Biochemical, Cleveland, Ohio) following themanufacturer's directions. The DNA and deduced amino acid sequences ofthe Hum4 V_(L) --UNIHOPE linker--FLAG peptide of pATDFLAG is shown inFIG. 28.

Generation of pSC49FLAG

The CC49V_(H) is inserted into the sites of Xho I - Nhe I pATDFLAG (seeFIG. 29) and evaluated for biological activity with the purpose ofserving as a positive control for the FLAG assay system to detectbinding to TAG-72. The new plasmid, called pSC49FLAG (see FIG. 29) isgenerated as follows. The plasmid pATDFLAG (5 mg, purified from a 2.5 mlculture by the Magic Miniprep™ system (Promega) is treated with Xho Iand Nhe I and the large vector fragment purified as described above forpSCFVUHH. The CC49 V_(H) insert DNA fragment is obtained by PCRamplification from pSCFVUHH and oligonucleotides UNI3 as the 5' endoligonucleotide and SC49FLAG as the 3' end oligonucleotide. Theresulting DNA and amino acid sequences of this SCFV antibody, with theFLAG peptide at the C-terminus, is shown in FIG. 30. The PCR reaction iscarried out using 100 pmol each of the oligonucleotides, 0.1 ng ofpSCFVUHH target DNA (uncut) and the standard protocol and reagentsprovided by Perkin Elmer Cetus. The DNA is first gel purified, thentreated with Xho I and Nhe I to generate sticky ends and purified from a4% polyacrylamide gel and electroeluted as described earlier. The DNAvector (pATDFLAG treated with Xho I and Nhe I) and the insert (CC49V_(H) PCR product from pSCFVUHH treated with Xho I and Nhe I) areligated in a 1:1 molar ratio, using 100 ng vector DNA (Stratagene kit)and used to transform E.coli AG1 competent cells (Stratagene) accordingto the manufacturer's directions. A colony with the correct plasmid DNAis picked as the pSC49FLAG clone.

Ligation of pATDFLAG Vector with PCR Amplified Human V_(H) Inserts

The protocol for the ligation reaction is as follows:

    ______________________________________                                        COMPONENT               AMOUNT                                                ______________________________________                                        DNA vector:pATDFLAG Xho 2.5 μL                                               I/Nhe I                                                                       Hum V.sub.H (X) DNA inserts: Xho 6 μL                                      I/Nhe I                                                                       10 mM ATP (Stratagene) 2 μL                                                10 × buffer (Stratagene) 2 μL                                        T4 DNA ligase (Stratagene) 1 μL                                            ddH.sub.2 O 6.5 μL                                                       ______________________________________                                    

DNA vector, ATP, 10×buffer and ddH₂ O are combined. DNA insert and T4DNA ligase are then added. Ligation reactions are placed in a 4 L beakercontaining H₂ O at 18° C. The temperature of the water is graduallyreduced by refrigeration at 4° C. overnight. This ligation reactiongenerates pHum4 V_(L) - hum V_(H) (X) (See FIG. 29).

Transformation of E.coli AG1 with pHum4 V_(L) -Hum V_(H) (X) LigationMix

Transformation of pATDFLAG into competent E.coli AG1 cells (Stratagene)is achieved following the supplier's protocol.

IBI MII Anti-FLAG Antibody Plate Assay

The first three steps, preparation of TAG-coated membranes, plating ofbacterial membranes, and assembly of TAG and bacterial membranes, arethe same as those described in the CC49-Biotin Competition Plate Assay.

After the 24 hour incubation at ambient temperatures, the membranes arewashed with TTBS three times at 250 rpm for four minutes. The MIIantibody (cat#IB13010, International Biotechnologies, Inc.) is thendiluted with TBS to a concentration ranging from 10.85 μg/ml to 0.03μg/ml. Ten milliliters of the diluted antibody are added to eachmembrane. The membranes are then incubated for 1 hour at ambienttemperatures and shaken on a rotary shaker at 70 rpm. After incubation,the MII antibody is removed and the membranes are washed three times at250 rpm and ambient temperatures for 5 minutes. The final wash isremoved and 20 milliliters of a 1:2000 dilution of sheep anti-mousehorseradish peroxidase linked whole antibody (cat#NA931, Amersham) isprepared with TBS and added to each membrane. The membranes are againincubated for 1 hour at ambient temperatures and 70 rpm. Followingincubation, the membranes are washed three times at 250 rpm and ambienttemperature for 5 minutes each. Enzygraphic Webs (cat# IB8217051,International Biotechnologies, Inc.) are used to develop the membranes,according to the manufacturer's instructions. The membranes are thenphotographed.

Instead of seeing a clear zone on the developed membrane for a positiveHum4 V_(L) -V_(H) (X) clone producing an SCFV that binds to TAG-72, (asseen with the competition screening assay) in this directFLAG--detecting assay, a blue-purple spot is indicative of a colonyproducing a SCFV that has bound to the TAG-72 coated membrane. Theadvantage of using the FLAG system is that any Hum4 V_(L) - V_(H) SCFVcombination that has bound to TAG-72 will be detected. Affinities can bemeasured by Scatchard analysis (Scatchard (1949), supra) and specificityby immunohistochemistry. These candidates could then be checked forbinding to a specific epitope by using the competition assay, supra, anda competing antibody or mimetic, if desired.

The present invention is not to be limited in scope by the cell linesdeposited since the deposited embodiment is intended for illustrationonly and all cell lines which are functionally equivalent are within thescope of the invention. Indeed, while this invention has been describedin detail and with reference to specific embodiments thereof, variouschanges and modifications could be made therein without departing fromthe spirit and scope of the appended claims.

What is claimed is:
 1. A Hum4 V_(L), V_(H) antibody which specificallybinds to TAG-72, or an antigen-binding fragment thereof, consistingessentially of at least one light chain variable region (V_(L)) and atleast one heavy chain variable region (V_(H)) wherein(a) the V_(L) is ahuman kappa Subgroup IV V_(L) encoded by the human Subgroup IV germlinegene (Hum4 V_(L)) or by one of the effectively homologous human kappaSubgroup IV genes or DNA sequences thereof, the V_(L) comprising humancomplementarity determining regions (CDRs) and human kappa Subgroup IVframework regions; (b) the V_(H) is an anti-TAG-72 V_(H) encoded by aDNA sequence encoding, as said V_(H), at least the heavy chain variableregion of an antibody which specifically binds TAG-72; and the V_(H) iscapable of combining with the V_(L) to form a three dimensionalstructure having the ability to specifically bind TAG-72.
 2. The Hum4V_(L), V_(H) antibody or fragment thereof of claim 1, wherein the V_(L)is further encoded by a human J gene segment.
 3. The Hum4 V_(L), V_(H)antibody or fragment thereof of claim 1 wherein the V_(H) is encoded bya DNA sequence which comprises the V_(H) αTAG germline gene or one ofits effectively homologous genes or productively rearranged derivatives.4. The Hum4 V_(L), V_(H) antibody or fragment thereof of claim 1,wherein the V_(H) is further encoded by a mammalian D gene segment. 5.The Hum4 V_(L), V_(H) antibody or fragment thereof of claim 1, whereinthe V_(H) is derived from the variable regions of CC46, CC49, CC83 orCC92.
 6. The Hum4 V_(L), V_(H) antibody or fragment thereof of claim 1,wherein the V_(H) comprises (I) CDRs encoded by a gene derived from theV_(H) αTAG, and (2) framework segments, adjacent to the CDRs, encoded bya human gene.
 7. The Hum4 V_(L), V_(H) antibody or fragment thereof ofclaim 1, wherein the light chain further comprises at least a portion ofa human constant region (C_(L)) and the heavy chain further comprises atleast a portion of a mammalian constant region (C_(H)).
 8. The Hum4V_(L), V_(H) antibody or fragment thereof of claim 7, wherein the C_(H)is human IgG1-4, IgM, IgA1, IgA2, IgD or IgE.
 9. The Hum4 V_(L), V_(H)antibody or fragment thereof of claim 7, wherein C_(L) is kappa orlambda.
 10. The Hum4 V_(L), V_(H) antibody or fragment thereof of claim1 wherein the antibody is MP1-44H produced by a cell line having theidentifying characteristics of ATCC HB 10426 or MP1-84H produced by acell line having the identifying characteristics of ATCC HB
 10427. 11. AHum4 V_(L), V_(H) antibody conjugate comprising the Hum4 V_(L), V_(H)antibody or fragment thereof of claim 1 conjugated to an imaging markeror a therapeutic agent.
 12. The Hum4 V_(L), V_(H) antibody conjugate ofclaim 11, wherein the imaging marker is selected from the groupconsisting of ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹¹¹ In, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga,¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, and ^(99m) Tc.
 13. The Hum4 V_(L), V_(H)antibody conjugate of claim 11, wherein the therapeutic agent is a drugor biological response modifier, radionuclide, or toxin.
 14. The Hum4V_(L), V_(H) antibody conjugate of claim 13, wherein the drug ismethotrexate, adriamycin or interferon.
 15. The Hum4 V_(L), V_(H)antibody conjugate of claim 13, wherein the radionuclide is ¹³¹ I, ⁹⁰ Y,¹⁰⁵ Rh, ⁴⁷ Sc, ⁶⁷ Cu, ²¹² Bi, ²¹¹ At, ⁶⁷ Ga, ¹²⁵ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁷⁷Lu, ^(99m) Tc, ¹⁵³ Sm, ¹²³ I or ¹¹¹ In.
 16. A composition for cancertreatment comprising a pharmaceutically effective amount of the Hum4V_(L), V_(H) antibody or fragment thereof of claim 1 in apharmaceutically acceptable, non-toxic, sterile carrier.
 17. Acomposition for cancer treatment comprising a pharmaceutically effectiveamount of the Hum4V_(L), V_(H) antibody conjugate of claim 12 in apharmaceutically acceptable, non-toxic, sterile carrier.
 18. Acomposition for cancer treatment comprising a pharmaceutically effectiveamount of the Hum4V_(L), V_(H) antibody conjugate of claim 13 in apharmaceutically acceptable, non-toxic, sterile carrier.
 19. The Hum4V_(L), V_(H) antibody or fragment thereof of claim 4, wherein the V_(H)is further encoded by a mammalian gene segment.
 20. A Hum4 V_(L), V_(H)single chain antibody which specifically binds to TAG-72, or anantigen-binding fragment thereof, consisting essentially of(a) at leastone light chain having a variable region (V_(L)), said V_(L) being ahuman kappa Subgroup IV V_(L), encoded by the human Subgroup IV germlinegene (Hum4 V_(L)) or by one of the effectively homologous human kappaSubgroup IV genes or DNA sequences thereof, the V_(L) comprising humanCDRs and human kappa Subgroup IV framework regions; (b) at least oneheavy chain having a variable region (V_(H)), said V_(H) being ananti-TAG-72 V_(H) encoded by a DNA sequence encoding, as said V_(H), atleast the heavy chain variable region of an antibody which specificallybinds TAG-72; and (c) at least one polypeptide linker linking the V_(H)and V_(L),wherein the V_(H) is capable of combining with the V_(L) toform a three dimensional structure having the ability to bind TAG-72 andthe polypeptide linker allows the proper folding of the V_(H) and V_(L)into a single chain antibody which is capable of forming saidthree-dimensional structure.